Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system

ABSTRACT

A fluid interface port in a microfluidic system and a method of forming the fluid interface port is provided. The fluid interface port comprises an opening formed in the side wall of a microchannel sized and dimensioned to form a virtual wall when the microchannel is filled with a first liquid. The fluid interface port is utilized to perform a labeling operation on a sample.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 10/028,852, filed Dec. 21, 2001 and entitledMicrofluidic System Including a Virtual Wall Fluid Interface Port forInterfacing Fluids With the Microfluidic System, the contents of whichare herein incorporated by reference. The present application alsoclaims priority to U.S. Provisional Patent Application Ser. No.60/391,872, filed Jun. 25, 2002 and entitled Microfluidic Chip withPost-Column Labeling, the contents of which are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to fluidic interface ports for providingbi-directional fluid interfacing in a microfluidic system. Morespecifically, the present invention relates to the use of a fluidicinterface port in a microfluidic system for performing a labelingoperation.

BACKGROUND OF THE INVENTION

In the chemical, biomedical, bioscience and pharmaceutical industries,it has become increasingly desirable to perform large numbers ofchemical operations, such as reactions, separations and subsequentdetection steps, in a highly parallel fashion. The high throughputsynthesis, screening and analysis of (bio)chemical compounds, enablesthe economic discovery of new drugs and drug candidates, and theimplementation of sophisticated medical diagnostic equipment. Of keyimportance for the improvement of the chemical operations required inthese applications are an increased speed, enhanced reproducibility,decreased consumption of expensive samples and reagents, and thereduction of waste materials.

Microfluidic devices and systems provide improved methods of performingchemical, biochemical and biological analysis and synthesis.Microfluidic devices and systems allow for the performance ofmulti-step, multi-species chemical operations in chip-based microchemical analysis systems. Chip-based microfluidic systems generallycomprise conventional ‘microfluidic’ elements, particularly capable ofhandling and analyzing chemical and biological specimens. Typically, theterm microfluidic in the art refers to systems or devices having anetwork of processing nodes, chambers and reservoirs connected bychannels, in which the channels have typical cross-sectional dimensionsin the range between about 1.0 μm and about 500 μm. In the art, channelshaving these cross-sectional dimensions are referred to as‘microchannels’.

By performing the chemical operations in a microfluidic system,potentially a number of the above-mentioned desirable improvements canbe realized. Downscaling dimensions allows for diffusional processes,such as heating, cooling and passive transport of species (diffusionalmass-transport), to proceed faster. One example is the thermalprocessing of liquids, which is typically a required step in chemicalsynthesis and analysis. In comparison with the heating and cooling ofliquids in beakers as performed in a conventional laboratory setting,the thermal processing of liquids is accelerated in a microchannel dueto reduced diffusional distances. Another example of the efficiency ofmicrofluidic systems is the mixing of dissolved species in a liquid, aprocess that is also diffusion limited. Downscaling the typicaldimensions of the mixing chamber thereby reduces the typical distance tobe overcome by diffusional mass-transport, and consequently results in areduction of mixing times. Like thermal processing, the mixing ofdissolved chemical species, such as reagents, with a sample orprecursors for a synthesis step, is an operation that is required invirtually all chemical synthesis and analysis processes. Therefore, theability to reduce the time involved in mixing provides significantadvantages to most chemical synthesis and analysis processes.

Furthermore, reduced dimensions enhance separation operations utilizedin chemical synthesis and analysis processes. One example is capillaryelectrophoresis, which is a separation technology based on the migrationof dissolved charged species through a liquid filled capillary by theapplication of a longitudinal electric field. By reducing thecross-sectional size of the capillaries, the separation efficiency cangreatly be improved, thereby resulting in rapid separations. Forexamples, see Effenhauser et al., Anal. Chem. 65:2637–2642 October(1993), Effenhauser et al., Anal. Chem. 66:2949–2953 September (1994),Jacobson et al., Anal. Chem. 66:4127–4132 December (1994) and Jacobsonet al., Anal. Chem. 66:1114–1118 April (1994).

Another aspect of the reduction of dimensions is the reduction ofrequired volumes of sample, reagents, precursors and other often veryexpensive chemical substances. Milliliter-sized systems typicallyrequire milliliter volumes of these substances, while microliter sizedmicrofluidic systems only require microliters volumes. The ability toperform these processes using smaller volumes results in significantcost savings, allowing the economic operation of chemical synthesis andanalysis operations. As a consequence of the reduced volume requirement,the amount of chemical waste produced during the chemical operations iscorrespondingly reduced.

It can be concluded that due to the reduced dimensions associated withmicrofluidic systems, important chemical operations can be acceleratedwhilst at the same instance lead to a reduction of consumption ofchemicals and chemical waste.

Applications of microfluidic systems are myriad. For example U.S. Pat.No. 5,922,591 describes a miniaturized integrated nucleic aciddiagnostic device and system. This device is capable of performing oneor more sample acquisition and preparation operations, in combinationwith one or more sample analysis operations. Useful applications formicrofluidic systems are in nucleic acid based diagnostics and de novosequencing applications. International Patent Appln. WO 96/04547,published Feb. 15, 1996, describes the use of electro-kinetic operatedmicrofluidic systems for performing electrophoretic separations, flowinjection analysis and chemical reactions and synthesis steps. U.S. Pat.No. 5,942,443 discloses a range of microfluidic devices and methods forperforming high-throughput synthesis and analysis, especially useful inscreening a large number of different chemical compounds for theireffect on a variety of chemical and biochemical systems. U.S. Pat. No.5,858,804 provides a method of performing an immunological assay in amicro-laboratory array comprising a plurality of microchannels andchambers disposed in a solid substrate. U.S. Pat. No. 6,176,991 B1discloses a serpentine electrophoresis channel in microchip formatproviding efficient, high-speed analysis of the composition of chemicalsamples, especially for nucleic acid sequencing.

Many methods have been described for the interfacing of fluids, e.g.,samples, analytes, reagents, precursors for synthesis and buffers,towards, within or between microfluidic systems. In conventionalmicrofluidic systems, the structures and methods used to introducesamples and other fluids into microfluidic substrates limit thecapabilities of the microfluidic systems. For example, conventionalmicrofluidic systems may include a separate sample introduction channelsfor introducing a sample to a microchannel for processing. The sample isfirst introduced into the sample channel and transported through thesample channel to the microchannel. Another method for introducing afluid involves the use of sample wells or reservoirs in communicationwith the microchannel for holding a relatively larger supply of thesample. Reservoirs are structures which accommodate a significantlygreater volume of fluid than the microfluidic channel. A relativelysmall portion of the sample supply in the sample well or reservoir isintroduced into the microchannel.

The total number of samples and other fluids that can be processed on amicrofluidic substrate is currently limited by the size and/or thenumber of reservoirs through which these fluids are introduced to themicrofluidic system. A disadvantage of known structures and methods forintroducing fluids into a microfluidic system is the use and transfer ofa much greater volume of fluid than is needed for microfluidic analysisdue to significant size inefficiencies and sample loss. Furthermore,with conventional methods of introducing fluids into microfluidicsystems, it is difficult to control the amount of sample introduces thatis eventually introduces into the microchannel after passing through asample channel or a reservoir.

One method of fluidic communication with microfluidic systems is bymechanical micropumps and valves, see U.S. Pat. No. 6,033,191 and U.S.Pat. No. 5,529,465. A major disadvantage of this approach for fluidicinterfacing is the complex construction and operation of thesemicropumps and valves. Another disadvantage is there relatively largesize and internal volume when compared to the internal volume of themicrochannels. Often there are multiple orders of magnitude betweenthese two volumes and the resulting discrepancy renders micropumpsunattractive to interface with a large number of small dimensionalmicrochannels.

U.S. Pat. No. 5,173,163 describes a method and device for introducing afluidic sample in a micro-capillary for electrophoretic separation. Inthis method liquid is brought into a separation capillary by introducingone end of this capillary into a vessel containing the liquid to beintroduced. A combination of applied external pressures and voltagesresults in the transport of liquid from the vessel into the capillary.The proposed method has disadvantages. The size of the device does notallow the interfacing with a large number of microchannels, and betweenconsecutive injections, the device needs to be cleaned, therebyconsiderably reducing throughput.

Another method for introducing materials in a microfluidic device isdisclosed in U.S. Pat. No. 6,042,709. In this approach electrokineticforces are employed to move a charged compound through a receiving inletopening of the microfluidic device. A disadvantage is that the preciseamount of injected liquids and substances depends upon a large number offactors which are difficult to control. One important parameter is thesurface potential of the microchannel wall, which together with theapplied voltage determines the liquid flow. This surface potentialdepends on pH of the liquid to be pumped as well as its ioniccomposition and even the type of ions present in the liquid. It is alsoa disadvantage that it does not allow the efficient interfacing with alarge number of different liquids as for every injection port, aseparate high voltage supply is required, together with the associatedliquid channels for providing a closed electrical circuit.

A method and apparatus for performing electrophoretic experiments in ahighly parallel fashion is disclosed in U.S. Pat. No. 6,103,199. Here, aplurality of separation capillaries with associated wells for receivingchemical substances in fluid form are disposed in the form of a twodimensional array. The chemical substances are dispensed from a microtiter plate into these wells by an interfacing methodology employingpressurized chambers associated with the wells to be filled. Adisadvantage is that only a very small fraction of the applied liquid isactually introduced in the target microchannel, the bulk of the appliedliquid drop remains behind in the well by capillary forces. As a result,most of the liquid is wasted and is not available for a consecutivechemical processing step. The effect that only a small portion of theliquid transported actually is introduced in a targeted part of amicrofluidic system, can be referred to as ‘injection efficiency’, i.e.the ratio between the volume of liquid required for a particularchemical operation in a part of the microfluidic system, and the totalvolume of liquid required for the introductory operation.

In this particular disclosure, only sub-nanoliter amounts are requiredfor the electrophoretic separation (i.e. the chemical operation), whilstmany microliters of sample are drawn from the micro titer plate (i.e.introductory operation), yielding an injection efficiency much less than0.001. A low injection efficiency is disadvantageous because itindicates inefficient use of chemical substances and an increasedproduction of waste.

U.S. Pat. No. 6,090,251 provides micro-fabricated structures forfacilitating fluid introduction into microfluidic devices. Fluid isintroduced into a plurality of receiving wells in direct communicationwith associated microchannels, by the dropping of liquids into thesereceiving wells using pressurized gas. Besides the complexity of therequired fluidic manifolds and pressurizing system, also here adisadvantage is the inherently low injection efficiency as only a verysmall fraction of the applied liquid is actually used in the experiment.

For the introduction of liquids in capillary electrophoresis columnsimplemented on chip-like devices, generally electrokinetic injection isapplied. See Woolley et al., Anal. Chem. 70:684–688 February (1998),Jacobson et al., Anal. Chem. 68:720–723 March (1996), Jacobson et al.,Anal. Chem. 66:2369–2373 July (1994) and Effenhauser et al., Anal. Chem.67:2284–2287 July (1995). In this method, liquid is pumped from a firstwell towards the microchannel for electrophoretic separation by theapplication of a high driving voltage between this well and a secondwell located downstream. Due to the charged inner surfaces of themicrochannel walls, an electroosmotic liquid flow is induced pumpingliquid out of the first into the targeted microchannel. This method isreferred to as ‘electrokinetic injection’ and has some specificdisadvantages. One disadvantage is that if a large number of liquidsneed to be handled, for instance in high-throughput synthesis andscreening applications, a large number of wells need to be integrated onthe microfluidic device. The relatively large footprint of a typicalwell (about 5 mm diameter) when compared to the microchannels in whichthe actual chemical operation is performed (about 50 μm diameter), takesup a dominating portion of the chip surface (see U.S. Pat. No. 6,143,152and U.S. Pat. No. 6,159,353). As the costs of microfluidic chipsstrongly depends on the chip surface, the required integration of wellsrenders this liquid injection scheme unattractive for high-throughputsynthesis and screening applications.

Another disadvantage of conventional systems is that for every well, aseparate electrode is required together with electronic circuitry forthe application of the driving voltages. This requirement results in acomplex and expensive apparatus.

Another specific disadvantage with electrokinetic injection is the factthat during the application of the driving voltage on the electrode theelectrolysis of water results in the generation of hydroxyl ions (OH⁻),hydrogen ions (H⁺), hydrogen gas (H₂) and oxygen gas (O₂). The generatedions will affect the acidity (i.e. pH) of the liquid pumped from thefirst well, whilst the produced gasses potentially give rise to theformation of gas bubbles in the microfluidic system thereby destroyingthe experiment and eventually the microfluidic device. Besideselectrolysis of the aqueous medium, any present electroactive speciescan degrade due to electrochemical reactions at the electrode surface.For instance, the presence of chloride ions, an ion present in mostliquid media, will result in the formation of chlorine gas, which onturn can interact and potentially destroy vulnerable (biochemical)compounds present in the liquid to be injected. In addition, the liquidto be injected can contain electroactive constituents, which can bedegraded by the electrochemical processes, associated withelectrokinetic injection. These disadvantages can be grouped andreferred to as ‘electrochemical pollution’.

Another disadvantage of electrokinetic injection is that in betweenconsecutive experiments, the well need to be thoroughly cleaned in orderto prevent cross-contamination. This required cleaning step results in areduction of throughput and makes it difficult to implement on-linemonitoring. Another disadvantage is that the liquid to be injected issubjected to a high voltage. This aspect of the absence of galvanicseparation makes it virtually impossible to use electrokinetic injectionfor in-vivo or near-vivo applications due to the danger ofelectrocution. Another disadvantage is that the electrokinetic injectionmethods referred to, are only applicable in chip like systems producedvia microfabrication technology, i.e. via the use of expensive equipmentand processes also applied for the fabrication of computer chips. Thesemethods are known to have high costs. It is desirable to provide an ninterfacing methodology, which is also applicable in current nonchip-based capillary systems. Still another disadvantage is the lowinjection efficiency of electrokinetic injection. To fill a typicalwell, about 10–50 μl of liquid is required, whilst for a particularchemical operation only sub nanoliter amounts are required.

U.S. Pat. No. 6,130,098 discloses the movement of liquid volumes intoand through microchannels by employing pressures generated by heating avolume of air in direct connection with these microchannels. Adisadvantage of this fluidic interfacing method is that for a correctand efficient operation the pressure generating air chamber togetherwith electronic heater components need to be integrated with themicrofluidic system. This results in a complex device with associatedlarge costs.

It can be concluded based on the above that current methods and systemsfor fluidic interfacing with microfluidic devices have particulardisadvantages regarding the difficulty of integration of a large numberof chemical operation nodes to interface with (i.e.electrochromatography columns, reactors etc.), relatively large requiredliquid volumes, low injection efficiency, electrochemical samplepollution, long rinse time between analysis steps, galvanic separationand required microfabrication technologies. Besides these disadvantages,none of the above mentioned interfacing methods allow bi-directionalfluidic interfacing, i.e. transporting liquids to and from microfluidicsystems. There has arisen a need in the art for providing suitablebi-directional fluidic interfacing structure that allows for theimplementation of a much wider range of chemical operation steps inmicrofluidic systems.

SUMMARY OF THE INVENTION

The present invention provides methods, devices and systems forinterfacing fluids in microfluidic systems. A fluid interface port fordirectly interfacing a microfluidic channel network and the surroundingenvironment is provided in a microchannel in a microfluidic system byforming an opening in a sidewall of the microchannel. The aperture formsa virtual wall when the microchannel is filled with a liquid. Theaperture has suitable cross sectional dimensions such that capillaryforces retain liquid within the microchannel. The virtual wall isdefined by the meniscus of the liquid in the opening, which essentiallyreplaces the sidewall of the microchannel so as to not substantiallyaffect or influence fluid flow through the microchannel.

Filling of a microchannel may be accomplished via the opening in theside wall of the microchannel. To fill the microchannel, a user formsdroplets of a first liquid and directs the droplet towards the opening,such that the droplets of the first liquid enter and fill themicrochannel.

Fluid introduction and/or fluid removal is accomplished through avirtual wall formed in the side wall of a microchannel. The process ofintroducing a fluid sample to the microchannel comprises forming adroplet of the fluid and propelling the droplet towards a virtual wall.The droplet traverses the virtual wall and enters the microchanneldirectly, without requiring intermediate reservoirs or channels. Thevirtual wall formed in the sidewall of the microchannel may also definea fluid ejection port for ejecting a fluid from the microchannel in theform of a droplet.

The present invention provides a system and method for performinglabeling operations on a sample. The labeling system employs a virtualwall as an interface port for one of the reactants in the labelingscheme. The system and method provide fast transient measurement ofunlabeled molecules based on their binding affinity and detection ofmolecules based on their binding affinity.

According to one aspect of the invention, a method of labeling a samplecomprises the steps of conveying the sample through a channel having afirst virtual wall fluid interface port and separating the sample in thechannel into a plurality of bands. The method further comprisesinjecting a labeling solution through the first virtual wall fluidinterface port, wherein the labeling solution interacts with one of thebands to form a labeled band.

According to another aspect of the present invention, a method oflabeling a sample comprises conveying the sample through a channelhaving a virtual wall fluid interface port and injecting a labelingsolution through the virtual wall fluid interface port. The labelingsolution interacts with the sample to label the sample.

According to yet another aspect of the present invention, a system forperforming a labeling operation comprises a column for conveying asample mixture through the system. The column comprises an interiorbounded by a side wall. The system further includes a separation regionfor separating the sample mixture into a plurality of bands and a firstfluid interface port downstream of the separation region for injectinglabeling molecules into the interior of the column.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a microfluidic system suitable forimplementing the illustrative embodiment of the invention.

FIG. 2 a is a top view of a microchannel structure having a fluidinterface port formed in a side wall of the microchannel for filling themicrochannel with a first fluid according to the teachings of theinvention.

FIG. 2 b is a cross-sectional view of the microchannel of FIG. 2 a alongthe length of the microchannel.

FIGS. 3 a and 3 b illustrate the steps of filling the microchannel ofFIG. 2 a with a first fluid by directing droplets of the first fluidthrough the fluid interface port.

FIG. 4 a illustrates the microchannel of FIGS. 2 a and 2 b furtherincluding a hydrophobic patch disposed in the microchannel interioraccording to an alternate embodiment of the present invention.

FIG. 4 b illustrates an alternate embodiment of a microchannel accordingto the teachings of the invention, wherein the hydrophobic patch iscoaxially arranged with a fluid interface port.

FIG. 5 a is a top view of a microchannel according to one embodiment,including a filling aperture and a stopper hole for directing thefilling of a first fluid in the microchannel according to the teachingsof the invention.

FIG. 5 b is a cross-sectional view of the length of the microchannelshown in FIG. 5 a, illustrating the process of filling the microchannel.

FIG. 6 illustrates the filling of a microchannel with two differentfluids by utilizing a plurality of filling apertures disposed in theside wall of the microchannel.

FIG. 7 a illustrates the process of closing the filling aperture ofFIGS. 2 a through 6 by forming and dispensing drops of encapsulant ontothe filling aperture according to one embodiment of the presentinvention.

FIG. 7 b illustrates the process of closing the filling aperture using acovering layer according to an alternate embodiment of the presentinvention.

FIG. 8 illustrates a microchannel having a filling aperture locatedadjacent to a closed end of the microchannel, such that the introductionof droplets into the microchannel through the filling aperture induces aflow in the microchannel.

FIG. 9 a is a cross-sectional side view of a microchannel including afluid interface port for receiving a liquid droplet into themicrochannel.

FIG. 9 b is a cross-sectional view perpendicular through themicrochannel of FIG. 9 a at the position of the fluid interface port,illustrating the insertion of a drop using a pin.

FIG. 9 c is a cross-sectional view of a microchannel having a virtualwall according to the teachings of the invention, illustrating thecomposition of the liquid inside the microchannel directly afterreceiving a droplet.

FIG. 9 d illustrates a microchannel having a virtual wall according tothe teachings of the invention, immediately after injection of thesecond liquid in the first liquid, whereby the first and second liquidare immiscible.

FIG. 9 e is a cross-sectional view of a microchannel having a virtualwall according to an alternate embodiment of the invention.

FIG. 9 f illustrates a microchannel having a conical fluid interfaceport forming a virtual wall according to another embodiment of theinvention.

FIG. 9 g is a cross-sectional view of a microchannel having a virtualwall according to an alternate embodiment of the invention, including acovering layer.

FIG. 9 h is a cross-sectional view of a microchannel having an array ofapertures forming virtual walls.

FIG. 9 i illustrates the introduction of a liquid into the microchannelshown in FIG. 9 h.

FIG. 10 a illustrates a microchannel having a plurality of virtual wallsdisposed across the radial width of the microchannel according to oneembodiment of the present invention.

FIG. 10 b illustrates a microchannel having a plurality of virtual wallsdisposed along the axial length of the microchannel according to oneembodiment of the present invention.

FIG. 11 a is a schematic block diagram representative of a sampleintroduction system for electrically guiding a selected droplet into aselected microchannel according to the teachings of the presentinvention.

FIG. 11 b is a cross-sectional view of a microchannel array of FIG. 4 aof the present invention wherein microchannels can selectively becharged to attract or repel a selected droplet according to theteachings of the present invention.

FIG. 11 c is a cross-sectional view of the microchannel array of FIG. 11a employing an array of ring electrodes for guiding a selected dropletinto a selected microchannel according to the teachings of the presentinvention.

FIG. 12 a is a schematic view of one embodiment of the present inventionfor ejecting liquid from a microchannel through a virtual wall by usingelectric fields.

FIG. 12 b is a schematic view of an alternate embodiment of the presentinvention for ejecting liquid from a microchannel through a virtual wallby the application of pressure pulses to the liquid.

FIG. 12 c is a schematic view of still another embodiment of the presentinvention for ejecting liquid from a microchannel through a virtual wallby the application of a gas pressure pulse to the liquid.

FIG. 12 d is a schematic view of yet another embodiment of the presentinvention for ejecting liquid from a microchannel through a virtual wallby a gas bubble.

FIG. 12 e is a schematic view of still another embodiment of the presentinvention for ejecting liquid from a microchannel through a virtual wallby the application of a gas pressure pulse to the liquid.

FIG. 12 f illustrates the ejector of FIG. 12 e in operation.

FIG. 12 g is a schematic view of an alternate means for ejecting liquidfrom a microchannel through a virtual wall using a pin assembly.

FIG. 13 a is a cross-sectional view of a microchannel with a singlevirtual wall wherein liquid inside the microchannel is opticallyverified via the virtual wall.

FIG. 13 b is a cross-sectional view of a microchannel in which twoconcentric virtual walls are diametrically opposed, and demonstratesoptical detection using both virtual walls.

FIG. 14 is a top view of an embodiment of the invention, illustratingthe use of a microchannel having a virtual wall to perform a separation.

FIG. 15 a illustrates a labeling system including a virtual wall formedin a microchannel according to an illustrative embodiment of theinvention.

FIG. 15 b illustrates a labeling system including a virtual wall formedin a microchannel according to an alternate embodiment.

FIG. 16 is a schematic diagram of a labeling operation according to anillustrative embodiment of the invention.

FIG. 17 shows a cutaway view of an embodiment of the invention in whicha plurality of virtual walls are employed for performing multi-stepchemical experiments.

FIG. 18 is a schematic cross-sectional view of an embodiment of thepresent invention in which a microchannel having a virtual wall part ofan electrokinetically operated microfluidic system.

FIG. 19 shows a perspective view of an embodiment according to theinvention in which a plurality of microchannels with virtual walls isapplied in an electrokinetically operated microfluidic system.

FIG. 20 illustrates an application of the virtual wall interface portsof an illustrative embodiment to interface a microfluidic system with amass spectrometer.

FIG. 21 a is an exploded view of a microfluidic chip manufacturedaccording to the teachings of the invention.

FIG. 21 b is a top view of the microfluidic chip of FIG. 21 a.

FIG. 21 c is a side view of the microfluidic chip of FIG. 21 a.

FIGS. 22 a–22 c illustrate the steps of manufacturing a microchannelhaving an opening suitable for forming a virtual wall according to anembodiment of the present invention.

FIGS. 23 a–23 c illustrate the steps of manufacturing a microchannelwith a virtual wall interface port according to another embodiment ofthe present invention.

FIGS. 24 a–24 c illustrate the steps of manufacturing a microchannelhaving a virtual wall as a fluidic interface port according to analternate embodiment of the present invention.

FIG. 25 a is a perspective view and of a microchannel with a virtualwall in which a hydrophobic patch is disposed in the microchannel.

FIG. 25 b shows the use of the microchannel with the virtual walldepicted in FIG. 25 a.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved fluidic interface forintroducing fluids to and removing fluids from a microchannel in amicrofluidic system. The invention further provides a method of formingthe fluidic interface. The present invention significantly improvescontrollability over fluid samples, increases injection efficiency andreduces waste by utilizing an opening defining a virtual wall in a sidewall of a microchannel to introduce and remove fluids.

One or more of the illustrative embodiments allow bi-directional fluidicinterfacing to and from microfluidic systems. With the presentinvention, a large number of chemical operations can be performed on asmall chip surface, thereby enabling the cost effective implementationand efficient operation of massively parallel synthesis and analysissystems. The present invention further significantly reduces therequired liquid volume for interfacing, resulting in a considerablereduction of consumption of chemicals as well as the reduction ofchemical waste. The present invention further provides methods andsystems for the injection of liquids with near 100% injection efficiencyand provides methods and systems for the injection of liquids which donot electrochemically pollute the handled liquids. The present inventionpresents methods and systems for the fast repetitive injection ofliquids in microfluidic systems, pressure driven as well aselectrokinetically driven, allowing high throughput synthesis, screeningand analysis applications. The present invention further allows forgalvanic separation of the liquid to be injected with theelectrokinetically operated microfluidic system. The invention furtherprovides fluidic interfacing with microfluidic systems devices andsystem that are not necessarily manufactured using standardmicrofabrication technologies. For example, the invention provides forinterfacing with standard fused silica capillaries. The presentinvention applies to a variety of liquid samples, including solutions ofcompounds, whole cells or cell lysates, enzymes, proteins or peptides,and particles.

FIG. 1 illustrates a microfluidic system suitable for implementing theillustrative embodiment of the present invention. The illustrativemicrofluidic system 10 comprises a substrate 11 having one or moremicrochannels 3 disposed therein. The microchannels transport fluidthrough the microfluidic system 10 for processing, handling, and/orperforming any suitable operation on a liquid sample. As used herein,the term “microfluidic” refers to a system or device for handling,processing, ejecting and/or analyzing a fluid sample including at leastone channel having microscale dimensions. The term “channel” as usedherein refers to a pathway formed in or through a medium that allows formovement of fluids, such as liquids and gases. The term “microchannel”refers to a channel preferably formed in a microfluidic system or devicehaving cross-sectional dimensions in the range between about 1.0 μm andabout 250 μm, preferably between about 25 μm and about 150 μm and mostpreferably between about 50 μm and about 100 μm. One of ordinary skillin the art will be able to determine an appropriate volume and length ofthe microchannel. The ranges are intended to include the above-recitedvalues as upper or lower limits. The microchannel can have any selectedshape or arrangement, examples of which include a linear or non-linearconfiguration and a U-shaped configuration. The microfluidic system 10may comprise any suitable number of microchannels 3 for transportingfluids through the microfluidic system 10.

According to one practice, the microchannel of the present invention caninclude a fluid interface port. As used herein, “fluid interface port”refers to a structure in a microfluidic system, such as an apertureformed in a microchannel, which provides fluid access between theinterior and the exterior of a microchannel. The fluid interface port isutilized to introduce fluids and other material to the microchanneland/or to remove fluid and/or other material from the microchannel. Thefluid interface port may comprise, among other applications, a fillingport for filling the microchannel with a carrier liquid, a sampleintroduction port for introducing a sample into the microchannel and anejection port for ejecting fluid from the microchannel.

According to the illustrative embodiment, the microchannel is defined bya side wall having any suitable shape enclosing at least a portion ofthe interior of the channel. The fluid interface port is formed in theside wall of the microchannel by removing a portion of the side wall todefine an opening. The fluid interface port of the illustrativeembodiment is formed by an aperture in the side wall of the microchannelhaving a diameter of between about 0.1 μm and about 200 μm andpreferably between about 25 μm and about 125 μm and most preferablybetween about 50 μm and about 100 μm. The aperture forming the fluidinterface port may have any suitable shape, including, but not limitedto, a cylinder, a disk, a conical shape, an elliptical shape and a cubicshape. The side wall or wall of the microchannel can be formed by two ormore components that bound the entire volume of the microchannel.According to a first aspect of the invention, the fluid interface portis utilized to fill the microchannel with a first liquid. The firstliquid comprises a carrier fluid for transporting a sample, reagent orother suitable liquid for performing one or more chemical operations,such as reactions and separations. Fluid flow may be induced in themicrochannel via any suitable means, including, but not limited to,pressurizing and electroosmotic means.

As illustrated in FIG. 2 a through FIG. 7, the fluid interface port 17formed in the sidewall of the microchannel forms a filling aperture ormicroaperture for filling the microchannel with the first liquid.Typically, conventional microchannels are filled by inserting the end ofthe microchannel into a reservoir containing the first liquid. Capillaryforces pull the first liquid into the microchannel and thereby fill themicrochannel with the first liquid. In order to avoid this procedure,the present invention employs a fluid interface port formed in thesidewall (not end) of the microchannel. This enables a user to fill themicrochannel through the port to provide simple, fast and efficientfilling without requiring a large, separate liquid reservoir.

FIG. 2 a is a top view of a microchannel structure 3 comprising a sidewall 16 surrounding a hollow interior. The microchannel includes a fluidinterface port 17 located in the side wall 16 of the microchannelstructure. The fluid interface port 17 may have any suitable shape basedupon the intended use, examples of which include circular, cylindrical,elliptical and conical. FIG. 2 b is a cross-sectional view of themicrochannel structure taken along the length of the microchannel 3,showing the fluid interface port 17 disposed in the top side of themicrochannel. As shown, the interior of the microchannel 3 is initiallyempty. According to the illustrative embodiment, the microchannel 3 isfilled with a first liquid via the fluid interface port 17 formed in theside wall 16 of the microchannel.

FIGS. 3 a and 3 b illustrate the process of filling the microchannelstructure 3 with a first liquid 4 by directing droplets 4 a of the firstliquid 4 toward the fluid interface port 17. The droplets 4 a enter themicrochannel 3 through the fluid interface port 17 and form a plug 13 ofthe first liquid 4 within the microchannel 3. As more droplets 4 a areintroduced via the fluid interface port 17, the plug 13 expands in bothdirections through the microchannel, as indicated by the arrows, therebyfilling the microchannel along the length of the microchannel with thefirst liquid 4. Moreover, capillary forces work to draw the liquid 4along the length of the microchannel 3. According to the illustrativeembodiment, the first liquid 4 forms a meniscus in the fluid interfaceport 17 when the microchannel is filled and the liquid is retained inthe microchannel by capillary forces.

The fluid interface port 17 is sized and dimensioned to form a virtualwall 15 when the microchannel is filled with the first liquid 4. As usedherein, “virtual wall” refers to the meniscus formed by the first liquid4 in the port 17 formed in the side wall of the microchannel. Themeniscus surface can be, although not required, substantially co-planarwith the wall 16 of the microchannel in which the meniscus is formed.The meniscus essentially replaces the removed portion of the side wallthat defines the aperture 17. The word “virtual” is chosen to expressthe effect that the overall liquid flow through the microchannel of themicrofluidic system is not influenced by the virtual wall, i.e. the flowof liquid in the microfluidic system having a virtual wall issubstantially identical to the flow of liquid through an identicalmicrofluidic system in which no virtual wall is present. The fluidinterface port, according to one practice, has appropriate dimensionsand surface properties as to substantially not influence the overallliquid flow and liquid shape when compared to a microfluidic system inwhich no port or meniscus is formed. The virtual wall forms a directinterface between the microchannel interior 5 and the microchannelexterior, allowing direct access to the liquid in microchannel 3 withoutintroducing dead or unswept volume in the microchannel 3. The virtualwall also serves to seal liquid inside of the microchannel through arange of pressures in the microchannel. Those of ordinary skill willreadily recognize that the surface or wall of the fluid interface portcan be formed anywhere along the axial height of the port. One ofordinary skill will recognize that the meniscus may be convex orconcave, depending on the appropriate system pressure.

According to an alternate embodiment, shown in FIG. 4 a, a hydrophobicpatch 23 is applied to the side wall or interior of the microchannel 3prior to introduction of the first liquid 4. When the first liquid 4 isintroduced to the microchannel, the hydrophobic patch 23 forms abarrier, and causes the plug 13 of liquid 4 to expand only in onedirection, away from the patch 23.

According to one embodiment, shown in FIG. 4 b, the hydrophobic patch 23is applied to the interior surface of the microchannel through anaperture forming a fluid interface port 17 in the sidewall 16 of themicrochannel. The hydrophobic patch 23 may comprise any suitablematerial for rendering a surface hydrophobic, such as gold. Thehydrophobic material may be sputtered, evaporated, sprayed or depositedthrough the aperture 17 and bound to the interior surface of themicrochannel 3 opposite the aperture to form the hydrophobic patch 23.One skilled in the art will recognize that any suitable surfacetreatment for the microchannel may be applied through the aperture, inaddition to hydrophobic patches.

The aperture used to apply the hydrophobic patch 23 may further serve asa vent for the microchannel 3 to allow air to escape from themicrochannel interior. As shown, the aperture is coaxially arranged withthe hydrophobic patch. The actual vent, formed by the aperture 17 in theside wall, is not hydrophobic. However, the microchannel surfacecoaxially opposite the vent is rendered hydrophobic by application ofthe hydrophobic patch 23. The hydrophobic region is not flushed byliquid, allowing air to escape the microchannel interior through theaperture 17 in the side wall 16.

According to yet another embodiment, shown in FIGS. 5 a and 5 b, themicrochannel 3 further includes a stopper hole 28 adjacent to the fluidinterface port 17. The stopper hole 28 is sized and dimensioned to forma pressure barrier for a liquid disposed in the microchannel. One ofordinary skill in the art will be able to determine a suitable size anddimension of the stopper hole to create the pressure barrier. Thestopper hole 28 acts as a stop for the meniscus of liquid filling themicrochannel, such that the liquid plug 13 formed by the first liquid 4extends only in one direction along the length of the microchannel 3,away from the stopper hole 28.

According to yet another embodiment, the cross-sectional dimensions ofthe microchannel 3 may be varied locally to affect the pressure withinthe microchannel interior. For example, the microchannel may be narrowedor widened at certain locations to increase or decrease the capillaryforces acting on a fluid in the microchannel interior. One of ordinaryskill in the art will be able to determine a suitable cross-sectionaldimension to achieve a desired pressure within the microchannelinterior.

Alternatively, as shown in FIG. 6, the microchannel 3′ includes twofluid interface ports 17 a and 17 b, two stopper holes, 28 a and 28 b,and a vent hole 8 disposed between the two fluid interface ports 17 aand 17 b to allow for filling of the microchannel 3′ with a plurality ofdifferent liquids. A first liquid 4 is introduced into the interior ofthe microchannel through the first fluid interface port 17 a. The firststopper hole 28 a causes the first liquid to extend along themicrochannel towards the vent hole 8. A second liquid 4′ is introducedinto the interior of the microchannel through the second fluid interfaceport 17 b and extends along the microchannel towards the vent hole 8,due to the presence and location of the second stopper hole 28 b. Air inthe microchannel is released through the vent hole 8.

After filling the microchannel via a fluid interface port 17, such as afilling hole disposed in the sidewall of the microchannel, the fluidinterface port may be closed to prevent leakage of the fluid from themicrochannel. According to the embodiment shown in FIG. 7 a, the fluidinterface port 17 is closed by dispensing drops of encapsulant 21 a ontothe fluid interface port 17. The encapsulant drops form a cap 21 andeffectively close the microchannel 3. Alternatively, as shown in FIG. 7b, the microchannel 3 may be closed after filling by adhering a closinglayer 41 to the microchannel 3 to cover the fluid interface port 17.

According to another embodiment, as shown in FIG. 8, the fluid interfaceport 17 may be utilized to provide a pumping mechanism to inducemovement of liquid through the microchannel. As illustrated, the fluidinterface port 17 may be disposed adjacent to a closed or open end ofthe microchannel 3. The introduction of droplets into the microchannelthrough the fluid interface port results in movement of liquid throughthe microchannel. This movement corresponds to the frequency of thedrops introduced to the hole. The movement of liquid induced by theintroduction of droplets through the filling hole can be applied in manymicrofluidic applications as an accurate pumping and/or dosingmechanism. Those of ordinary skill will readily recognize that themicrochannel can employ a hydrophobic patch or a stopper hole to induceliquid movement in a selected direction or any suitable means forconveying liquid through the microchannel.

According to another aspect of the invention, a second fluid sample canbe introduced to the microchannel 3 through the same or different fluidinterface port 17, after the microchannel is filled with the firstliquid 4. FIG. 9 a is a side view of a microchannel 3 according to anillustrative embodiment of the invention including a fluid interfaceport 17 formed in the side wall 16 of the channel and showing a dropletgenerating system 18 for forming droplets of the second fluid sample tobe introduced into the microchannel 3. The fluid interface port 17provides a direct interface between the microchannel interior and theexterior. According to the present invention, the interface between afirst liquid 4 in the microfluidic system and a surrounding gas phase isdefined by the local absence of a solid wall in the microchannel, ratherthan a separate channel or reservoir structure.

According to one aspect, the virtual wall 15 formed in the port 17 canbe used as a sample introduction port for introducing the second liquidinto the first liquid 4 in the form of liquid droplets. A second liquid19 a can be directly injected into the first liquid 4 withinmicrochannel 3 through the virtual wall 15 without requiring anintermediate structure, such as a sample introduction channel or asample reservoir. According to the illustrative embodiment, the secondliquid is introduced by forming a droplet 19 b of the second liquid 19 aand directing the droplet towards the virtual wall 15 with anappropriate speed and direction, indicated in FIG. 9 a by velocityvector V, so as to traverse the virtual wall 15 and enter the interiorof the microchannel.

The liquid droplets may be formed and dispensed using any suitabledroplet forming system 18, such as the droplet dispensing systemsdescribed in U.S. Provisional Patent Application Ser. No. 60/325,001filed Sep. 25, 2001 and entitled “Two-Pin Liquid Sample DispensingSystem”, the contents of which are herein incorporated by reference, andU.S. Provisional Patent Application No. 60/325,040 entitled “DropletDispensing System”, the contents of which are herein incorporated byreference.

According to the illustrative embodiment, the lateral dimensions of thefluid interface port 17 are substantially identical to or less than thediameter of the microchannel 3, whilst the diameter of the illustrateddroplet 19 b is smaller than the lateral dimensions of the fluidinterface port 17. The fluid interface port 17 has a dead volume that issubstantially smaller when compared to conventional fluid interfaceports, such as a well or a sample introduction channel. As used herein,“dead volume” refers to the volume of liquid retained in the fluidinterface port 17 (i.e. the volume of liquid the fluid interface portholds that is not flushed through the fluid interface port by the flowfield of the first liquid 4 through the microchannel). The total volumeof the fluid interface port 17 is defined by the area of the apertureformed in the side wall and the thickness of the sidewall 16. The volumeof the first liquid 4 filling the fluid interface port defines the deadvolume. According to the illustrative embodiment, the fluid interfaceport has a dead volume that is less than about one nanoliter andpreferably less than one picoliter, and most preferably about zero.Preferably, the dead volume is less than the volume of liquid samplethat is injected through the fluid interface port 17.

The size of the aperture and the hydrophobicity of the fluid interfaceport determine the size of the dead volume. For example, themicrochannel shown in FIG. 9 a has zero dead volume i.e. no liquid isretained in the fluid interface port 17 and a sample injected throughthe port 17 directly enters the microchannel interior. According toother embodiments, the first liquid may partially or totally fill theaperture, and the dead volume may be a non-zero, but substantiallysmall, value. The dead volume also depends in whether the meniscus 15bulges up or down, a factor that is controlled by the hydrophobicity ofthe port 17, the properties of the liquid filling the microchannel 3 andthe size of the aperture forming the port 17.

The relatively small dead volume provided by the virtual wall 15 resultsin a direct fluid interface allowing direct injection of a precisevolume of sample into the interior of the microchannel 3 from theexterior of the microchannel. The ability to directly inject sample intothe microchannel due to the low dead volume of the fluid interface port17 provides improved control over the amount of sample that is injectedinto the microchannel, allows efficient use of sample, and significantlyreduces waste of the sample. Furthermore, the direct injection providedby the very small dead volume reduces or prevents cross-contaminationbetween different samples and allows a third liquid to be directlyinjected into the system immediately after a second liquid withoutrequiring flushing of the fluid interface port. Conversely, inconventional microfluidic systems employing a sample introductionchannel, sample well or sample reservoir for introducing a fluid sampleto a microchannel, the dead volume is significantly large relative tothe size of the microchannel. In order to introduce a fluid sample intothe microchannel interior, the fluid sample must first pass through thedead volume. A larger dead volume leads to dispersion of the sample, atime delay between the time of injection and the time when the sampleenters the microchannel, injection inefficiency, potentialcross-contamination between different samples and difficulty controllingthe amount of sample that actually reaches the microchannel. Theseproblems are avoided or reduced by the use of the fluid interface port17 forming a virtual wall 15 according to the illustrative embodiment.

FIG. 9 b shows a cross-sectional view perpendicular to the microchannel3 at the location of fluid interface port 17, illustrating the processof introducing the second liquid 19 a into the first liquid 4 throughthe virtual wall 15. As illustrated in FIG. 9 b, the droplet generatingsystem 18 comprises a droplet carrying element for carrying the droplet.According to the illustrative embodiment, the droplet carrying element180 comprises a pin, as described in U.S. Provisional Patent ApplicationSer. No. 60/325,001 filed Sep. 25, 2001 and entitled “Two-Pin LiquidSample Dispensing System”, and U.S. Provisional Patent Application No.60/325,040 entitled “Droplet Dispensing System”, for introducing thedroplet to the aperture by contacting the virtual wall 15.

FIG. 9 c shows a cross-sectional view of the microchannel 3 immediatelyafter injection of the second liquid 19 a in the first liquid 4. Asillustrated, the second liquid 19 a forms a well defined plug 14 in thefirst liquid 4. According to an alternate embodiment, the second liquiddissolves, merges or mixes into the first liquid. After introduction viathe virtual wall, the second liquid 19 a is transported through themicrochannel by the first liquid 4.

According to one embodiment of the invention, illustrated in FIG. 9 d,the second liquid, which is introduced into the microchannel 3 via thevirtual wall, is immiscible with the first liquid. FIG. 9 d shows across-sectional view of the microchannel 3 immediately after injectionof the immiscible second liquid 19 a in the first liquid 4. Afterpenetrating the virtual wall 15, a substantially spherical, micelle-likeliquid volume 71 of the second liquid is formed in the first liquid 4.As both liquids are immiscible with each other, this liquid volume 71remains confined and can be separately transported along themicrochannel 3.

The illustrative mode of liquid injection into a microfluidic system viaa virtual wall and consecutive handling is particularly preferable forperforming a liquid-liquid extraction. Liquid-liquid extraction is awell-known technique, widely used in many chemical analysis andsynthesis steps, especially during drug discovery. In liquid-liquidextraction, two immiscible liquids are brought in intimate contact toallow the extraction of specific components from the liquid containing aspecific substance (i.e. the source liquid) into the extraction medium(i.e. the target liquid). A suitable example of liquid-liquid extractionis the extraction of a water-soluble substance synthesized in an organicsolvent, whereby said solvent is immiscible with water. The organicsolvent containing the water-soluble synthesis product of interest ismixed with water to form small micelle-like droplets of solvent in thewater phase. The water soluble substance diffuses out of the solvent andis collected in the water phase. The water phase can be re-collectedafter a predetermined time, due to the immiscible properties of the twosubstances. In this manner, the water soluble substance is thusextracted from the organic liquid phase. Of key importance is the totalcontact surface between both liquids. A larger exchanging surface,provided by the shape of the droplets, results in a faster extractionprocess.

The microfluidic system of the present invention may be utilized toperform a liquid-liquid extraction between a water soluble substance andan organic liquid phase, which is immiscible with water, as describedbelow. To perform a liquid-liquid extraction between a water solublesubstance and an organic liquid phase, the illustrative microchannel 3is filled with a suitable first liquid 4, which is, according to theillustrative embodiment, an appropriate aqueous solution. The firstliquid 4 forms a virtual wall 15 at the opening 17 disposed in thesidewall of the microchannel. Droplets 19 b of the organic phasecontaining the substance are formed and injected into the first liquid 4through the virtual wall 15. After the droplets 19 b traverse thevirtual wall, micelle-like liquid volumes 71 are formed in themicrochannel 3, which, due to their very small size, have a relativelyvery large exchange surface area. Consequently, the water solublesubstance initially present in the micelle-like liquid volumes 71 isextracted into the first liquid 4 and is available for furtherprocessing. To concentrate the substance in a consecutive step, theextracted micelle-like liquid volumes 71 are separated from liquid 3 bya suitable separating technique as known in the art, including, but notlimited to, electrophoresis, dielectrophoresis, gravitational or bodyforces, capillary forces and special selective sieves.

According to an alternate embodiment, shown in FIG. 9 e, the fluidinterface port 17 disposed in the microchannel side wall 16 and forminga virtual wall 15 may have any suitable shape, such as a cylindrical orconical shape, having a suitably low dead volume for providing directaccess to the microchannel interior. According to one embodiment, theinner wall 63 of the fluid interface port 17 is formed of or coated witha material that is repellant for the first liquid 4 to repel the firstliquid from the opening 17. According to a preferred embodiment, theinner wall 64 of the microchannel 3 is attractive for the first liquid4, to retain the first liquid inside the microchannel. The liquidrepellent section in the fluid interface port 17 prevents liquid fromleaking out of the microfluidic system and ensures the repeatableformation of a virtual wall 15 in the fluid interface port 17 when themicrochannel is filled with liquid. The use of an inner wall 64 that isattractive for the first liquid 4 further enhances automatic, passivecapillary filling of the microchannel 3 via the port 17, as describedabove, or by providing the first liquid 4 at one end of the microchannel3. As a result of capillary forces, the microchannel 3 may beautomatically filled without requiring application of external energy orpressure sources, such as pumps or pressure chambers.

As shown in FIG. 9 f, the fluid interface port 17 may have an invertedconical shape to facilitate formation of the virtual wall when thecorresponding microchannel 3 is filled with liquid. For example, theshaft forming the fluid interface port 17 may have a height H of about0.175 mm, with an aperture at the bottom having a diameter D1 of betweenabout 0.05 mm and about 0.2 mm, preferably about 0.1 mm, and an aperturein the top of the fluid interface port 17 having a diameter D2 ofbetween about 0.2 and 0.3 mm, and preferably about 0.25 mm. As shown,the meniscus, defining the virtual wall 15, is formed at the bottom ofthe shaft and has a diameter of about 0.1 mm upon filling of themicrochannel 3.

FIG. 9 g illustrates an embodiment where the microchannels and virtualwall 15 are covered with a covering layer 66. According to theillustrative embodiment, the covering layer 66 comprises a liquid layerthat is immiscible with the first liquid 4 in the microchannel. Thecovering layer prevents the evaporation of the first liquid 4 from themicrochannel through the opening 17, while still allowing the injectionof a second liquid, such as liquid 19 a, into the microchannel throughthe covering layer 66 and the virtual wall 15.

According to an alternate embodiment, as shown in FIG. 9 h, the fluidinterface port in the microchannel is formed by an array 72 of openings17, each forming a virtual wall 15 upon filling of the microchannel 3with the first liquid 4. The virtual walls 15 in the array 72 aredisposed in close proximity to each other, thereby allowing theinjection of liquid via a wicking process, as illustrated in FIG. 9 i.To introduce a second liquid into the microchannel, as shown in FIG. 9i, a selected amount of the second liquid 19 a is deposited on top ofthe array 72, such that the capillary forces wick the second liquid intothe microchannel 3. According to a preferred embodiment, the inner walls63 of the fluid interface port 17 are rendered repellant to the firstliquid 4 whilst the outer surfaces 65 of the fluid interface ports 17preferably are rendered attractive to the second liquid 19 a. The use ofan array of openings to form an array of virtual walls reduces thenecessity and criticality of precisely targeting the droplets 19 btowards a particular virtual wall. The droplets need only to be aimed inthe direction of the array, allowing capillary forces to pull dropletsinto the channel interior. The velocity and direction of the propelleddroplets are also not as important to achieve injection of the sampleinto the microchannel 3.

According to yet another embodiment of the invention, a plurality ofopenings are disposed in the sidewall of the microchannel to allow forthe introduction or ejection of liquid via a virtual wall at a pluralityof locations in the microchannel. For example, as shown in FIG. 10 a,the microchannel can include multiple fluid interface ports 17 a, 17 bpositioned across or along the width of the microchannel 3 to define aplurality of virtual walls to allow for simultaneous introduction of aplurality of liquids. In this manner, an increased volume of liquid maybe immediately injected into the microchannel 3 via the plurality ofvirtual walls. The use of a plurality of virtual walls across themicrochannel width further allows for simultaneous introduction andmixing of a plurality of different liquids. As shown in FIG. 10 a, themicrochannel includes a first liquid 4. A second liquid 19 a may beintroduced via a first virtual wall 15 a. Simultaneously, a third liquid190 a may be introduced via a second virtual wall 15 b. The secondliquid 19 a and the third liquid 190 a mix together with the firstliquid 4, as illustrated by the diffusion profiles of the liquids.

Alternatively, as shown in FIG. 10 b, the microchannel 3 may include aplurality of virtual walls disposed along the length of the microchannelto allow for sequential introduction of liquids into the microchannel,or ejection of a liquid from the microchannel along different locationsin the fluid flow path.

The illustrative microfluidic system 10 employing a virtual wall 15 in afluid interface port may be utilized with a sample introduction systemfor forming and guiding a droplet into a microchannel via a virtualwall. FIG. 11 a illustrates the microfluidic system 10 employing asuitable sample introduction system 67 for guiding a charged droplet 19d of a selected sample into a microchannel 3 a of selected interest. Theillustrated sample introduction system 67 includes a droplet generator18 for forming droplets of a selected liquid, a droplet charging circuit53 for selectively charging a droplet, and a droplet guiding system 150,comprising a ground electrode 54 and a plurality of electricallycontrolled deflection plates 56, 57, for establishing an electrostaticfield to direct the charged droplets to a selected location. Theillustrative microfluidic system 10 can be coupled to electronics forcontrolling the formation and guidance of the droplet to a selectedmicrochannel.

The droplets are generated by the droplet generator 18 having a nozzleassembly 70. The nozzle assembly 70 ejects liquid and forms individualdroplets of the liquid at a breaking off point 62. The droplet chargingcircuit 53 is pre-programmed and a corresponding electrode 52 ispositioned within the nozzle assembly to charge a pre-selected dropletwith either a positive or negative charge at the breaking off point 62.Surrounding the breaking off point 62 of the droplet formation is aground electrode 54. After breaking off and passing the ground electrode54, the charged droplet 19 c travels through an electrostatic fieldestablished by the first electrically controlled deflection plate 56 andthe second electrically controlled deflection plate 57. A plate-chargingcircuit 55 is associated with each plate and controls the polaritiesthereof to provide proper electrical charging to the respectivedeflection plates. As shown in FIG. 11 a, the droplet 19 d is chargedpositively and is deflected towards a virtual wall 15 disposed in theselected microchannel 3 a by negatively charging the first plate 56 andpositively charging the second plate 57. By controlling the charge onindividual droplets with droplet charging circuit 53 as well ascontrolling the electric field between the first plate 56 and the secondplate 57, droplets can be guided and effectively targeted to aparticular channel to effect pre-programmed chemical experiments.

FIG. 11 b is a cross-sectional view of a plurality of microchannels 3with associated fluid interface ports 17 employing a different chargingtechnique according to an alternate embodiment of the present invention.In the embodiment shown in FIG. 11 b, the droplet guiding system 150comprises an electrode 58 for each microchannel in the system and achannel charging circuit 59 for charging the microchannels. Eachmicrochannel 3 includes a corresponding electrode 58 for enhancedtargeting of the droplets by selectively charging the correspondingmicrochannel. The electrode 58 is connected to the channel chargingcircuit 59, associated with the droplet charging circuit 53 shown inFIG. 11 a, for generating a selected charge in each of the microchannels3. The channel charge interacts with the charged droplets to guide orsteer the charged droplet 19 d towards a respective microchannel 3 a. Asseen in FIG. 11 b, the droplet 19 d is positively charged whilst aselected microchannel 3 a is charged negatively, thereby resulting in anattractive force between the positively charged droplet 19 d and thevirtual wall 15 of the negatively charged microchannel 3 a. Guiding isenhanced by charging neighboring microchannels 3 b and 3 c withidentical sign as the selected droplet 19 d, which consequently repelthe positively charged droplet 19 d towards the selected microchannel 3a.

FIG. 11 c shows an alternate approach to enhance targeting of a chargeddroplet 19 d into a selected microchannel 3 a of a plurality ofmicrochannels 3, according to the present invention. In the embodimentshown in FIG. 11 c, the droplet guiding system 150 comprises a pluralityof targeting electrodes 61, each associated with a corresponding fluidinterface port or microchannel. The fluid interface ports andcorresponding microchannels are charged by a targeting electrodecharging circuit 61 to result in a force towards the virtual wall 15 ofthe selected microchannel 3 a. As seen in FIG. 11 c, the selecteddroplet 19 d is positively charged whilst the targeting electrode 61associated with the selected corresponding microchannel 3 a is chargednegatively to guide the selected droplet 19 d into a selectedcorresponding microchannel 3 a via the virtual wall 15. Guiding isenhanced by charging a neighboring targeting electrode 61 with anidentical sign as the selected droplet 19 d to repel the droplet awayfrom the neighboring microchannels 3 b, 3 c and towards the selectedmicrochannel 3 a.

According to an alternate embodiment of the invention, the dropletguiding system 150 may comprise a machine vision system that can employfiducial marks on one or more components of the system. One skilled inthe art will recognize that any suitable droplet guiding system fordirecting a droplet towards a virtual wall may be utilized in accordancewith the teachings of the invention.

According to the present invention, the fluid interface port 17 of themicrofluidic system 10 can optionally be utilized as a bi-directionalfluidic interface for the microchannel. In addition to providing aninterface for introducing a sample to a microchannel, the illustrativevirtual wall formed in the microchannel 3 filled with the first liquid 4may also be utilized as an ejection port for ejecting fluid from themicrochannel. FIGS. 12 a through 12 d show a cutaway view of amicrochannel 3 in which a fluid interface port 17 disposed in a sidewallof the channel can be used as an ejection port. The port 17 forms avirtual wall to allow the ejection of the first liquid 4 frommicrochannel 3. A suitable ejector 108 is provided in communication withthe microchannel to effect ejection of the first liquid through thevirtual wall 15. One skilled in the art will recognize that the ejector108 may comprise any suitable device or system for ejecting a dropletfrom the microchannel via the virtual wall fluid interface port.

According to the illustrated embodiment shown in FIG. 12 a, the ejectorcomprises a first electrode 34 disposed in the first liquid 4 and anelectrospray electrode 32 positioned in the vicinity of the port 17. Avoltage generator 31 is connected to the first electrode 34 and theelectrospray electrode 32 for applying a potential difference andgenerating an electric field between the electrospray electrode 32 andthe first liquid 4. The electric field generated by the voltagegenerator 31 between the electrodes results in an attractive force onthe first liquid 4, thereby effectively pulling the first liquid 4 outof the microchannel through the virtual wall 15 in the form of a droplet33. The virtual wall and microchannel arrangement shown in FIG. 12 a canalso be used to receive droplets of a second liquid through the virtualwall, as discussed above, by removing the electric field, therebyachieving a bi-directional interface.

FIG. 12 b shows another embodiment of the microfluidic system 10 of thepresent invention suitable for ejecting the first liquid 4 from themicrochannel 3. According to this embodiment, the ejector comprises apressure pulse generator 51 in communication with the first liquid 4 inthe microchannel 3. The pressure pulse generator 51 applies a pressurepulse having a selected amplitude, frequency and duration to the fluid 4to effectively eject the first liquid 4 through the virtual wall to forma droplet 33. The pressure pulse generator 51 can be any suitablestructure for generating a pressure pulse within the microchannel 3 andcan include a piezoelectric element, an electromagnetic actuator, aheater, an electrostatic actuator, comprising electrodes that move underthe influence of an applied voltage, an electrophoretic pressureactuator, comprising electrodes in channel on which a voltage pulse isapplied, pressurized gas or any other suitable pressure pulse generator.

The pressure pulse generator 51 can either be integrated at least partlywithin the microchannel 3 or can be fully external and working with thechannel, such as a rod-like moving actuator tapping locally on thechannel.

According to another embodiment, shown in FIG. 12 c, a gas pressurizer51 a is utilized to eject the liquid from the microchannel. According tothe illustrative embodiment, the gas pressurizer 51 a comprises apressurized gas tank, which applies pressure via a fast valve. A secondfluid interface port 17 b forming a second virtual wall 15 b can beformed in the microchannel 3. The second virtual wall 15 b issubstantially coaxially aligned with the first virtual wall formed inthe first fluid interface port 17. The second, coaxial virtual wall 15 bis in communication with the gas pressurizer 51 a which generates a gaspressure pulse having a selected amplitude, frequency and duration as toeffectively eject the first liquid 4 through the first virtual wall 15in the form of a droplet 33. During the ejection process, the secondvirtual wall 15 b is displaced inwards towards the first liquid 4 so asto form the droplet 33.

According to an alternate embodiment, as shown in FIG. 12 d, the ejectorcomprises a heater 51 b disposed in the microchannel 3 along the sidewall. The heater 51 b locally heats the first liquid 4 in order to forma droplet. The heating of the first liquid results in a quickly growinggas vapor bubble 51 c, which effectively ejects the droplet 33 from themicrochannel 3 via the virtual wall 15. The heater may comprises aheated spot, an electrical heater, an optically induced heater or anyother suitable heater.

According to one embodiment, shown in FIGS. 12 e and 12 f, thepressure-pulse generator 51 is formed in a holder 500 for a microfluidicchip 10 containing the microchannel 3. The pressure pulse generator canbe any suitable device for applying a pressure, such as a piezo-actuatedmembrane. According to one practice, a membrane 510 is connected to aconical-shaped pressure chamber 511, which is placed opposite thevirtual wall 15 and communicates with the microchannel interior. Thepressure pulse generator 51 may further include a through-hole 515formed in the holder and an O-ring 512 for sealing the pressure chamber511. To eject droplets from the microchannel, as shown in FIG. 12 d, anexternal voltage source applies voltage pulses (typically 500microseconds/200 V) to form a series of droplets 33 of about tennanoliters in volume. The illustrative pressure-pulse generator 51 iscapable of achieving ejection frequencies of up to 25 Hz, though oneskilled in the art will recognize that the invention is not limited tothis range. Alterations to the pressure-pulse generator 51 may be madein accordance with the invention. For example, the pressure-pulsegenerator 51 is not required to form part of chip holder 500 and may beintegrated on the microfluidic chip 10 itself.

According to yet another embodiment, shown in FIG. 12 g, the ejector 108comprises a dedicated spotting pin assembly 108 for removing a selectedamount of liquid from the microchannel 3 via the fluid interface port17. The illustrative pin assembly 1080 allows selective removal ofsub-nanoliter liquid volumes, though one skilled in the art willrecognize that the invention is not limited to this range. According tothe embodiment shown in FIG. 12 g, the dedicated spotting pin 1080 maycomprise a micromachined silicon spotting pin system comprising two pins1080 a and 1080 b that are spaced a predetermined distance apart. Thespace between the pins forms an open capillary 1081, which isdimensioned to accurately pick up predetermined amounts of liquid. Toremove a liquid volume from the microchannel 3, the pin is dippedthrough the port 17 into the microchannel 3. The pin 1080 automaticallydraws liquid into the capillary 1081 via capillary action. The pin 1080is then removed from port 17 and the capillary force produced betweenthe surfaces of the pins 1080 a and 1080 b holds the droplet in thecapillary 1081 formed between the two pins 1080 a and 1080 b. Accordingto the illustrative embodiment, the pin assembly 1080 is able ofremoving a liquid volume of 300 picoliters from the microchannel, whichcan be then spotted on a glass slide for further analysis or storage.The illustrative pin assembly 1080 allows a sampling time of about 1second or less.

A suitable pin assembly 1080 is described in U.S. patent applicationSer. No. 10/027,171, filed Dec. 21, 2001 and entitled “MicrofabricatedTwo-Pin Liquid Sample Dispensing System” the contents of which areherein incorporated by reference.

According to yet another embodiment of the invention, the virtual wall15 formed in the sidewall 16 of the microchannel 3 is utilized tooptically analyze the interior of the microchannel. FIG. 13 a shows anembodiment of the invention where the first liquid 4 disposed in themicrochannel is optically inspected by a light beam 27 focused by anoptical element 26. The light beam 27 is co-axially aligned with thefluid interface port 17 in the sidewall as to directly penetrate thefirst liquid 4 without impinging upon the channel wall 16 opposite theport 17. The optical element 26 can be any suitable lens or prism. Asuitable detector 68 is disposed adjacent to the virtual wall 15 tomonitor and analyze the liquid in the microchannel 3. In an alternateembodiment, a plurality of fluid interface ports 17 and 17 b are atdisposed in the side wall at right angles to each other and scattering,rather than absorption, is analyzed.

According to an alternate embodiment for optically analyzing the firstliquid 4 through the virtual wall 15, a second virtual wall 15 b isdisposed in the microchannel 3. FIG. 13 b shows an embodiment foroptical analysis of first liquid 4 in microchannel 3, in which a firstfluid interface port 17 forming a first virtual wall 15 is disposed onone side of the microchannel 3 and a second fluid interface port 17 bforming a second virtual wall 15 b is disposed on the opposite side ofmicrochannel 3, such that the first virtual wall 15 and the secondvirtual wall 15 b are substantially co-axially arranged. The light beam27 passing through and focused by the optical element 26 issubstantially co-axially positioned relative to the first virtual wallsuch that the light beam 27 directly penetrates the first liquid 4through the port 17, and after traveling through the first liquid 4exits on the opposing side of the microchannel via the second virtualwall 15 b. The light passing through the second port 17 b can becollected, focused or collimated by a second optical element 26 a incommunication with an optical detector 68 to monitor and analyze theliquid in the microchannel 3. Such optical detection means are ideallysuitable for analyzing liquids containing compounds that areintrinsically colored or fluorescent, or that are labeled with anoptically detectable moiety as described elsewhere herein.Alternatively, mass spectrometry may be used as a detection means when amicrofluidic chip of the present invention is interfaced with a massspectrometer.

According to one application of the present invention, a microfluidicsystem employing an aperture sized and dimensioned to form a virtualwall as a fluid interface port may be utilized to purify or filter asample. For example, the illustrative arrangement may be utilized topurify a DNA sample by separating contaminants from the DNA fragments inthe sample. As shown in FIG. 14, a microchannel 3 having a fluidinterface port 17 formed in the sidewall and defining a virtual wall isconnected to a plurality of waste channels 136 and an outlet channel 137formed at the intersection of the waste channels 136 and themicrochannel main body 3. The microchannel 3 has an inlet 138 having asmaller diameter than the main body of the microchannel. Themicrochannel 3 is filled with a suitable washing medium 4 to effectseparation of a sample that is introduced via the virtual wall.

To achieve separation of a sample, such as the separation of a DNAsample from contaminants, a fluid flow is induced in the washing mediumthrough the microchannel and the sample is injected into the flowingwashing medium via the virtual wall 15. The arrangement exploitsdiffusion to separate the different components of the sample. The largermolecules (i.e. the DNA fragments) in the sample and the smallermolecules in the sample (i.e. the contaminants) diffuse in the washingmedium with different diffusion rates, which effectively separates thedifferent components of the sample according to size. For example, thesmaller molecules diffuse into the washing medium faster than the largermolecules. The two sample streams, the residual sample stream containingthe larger particles and the diffused sample stream containing thecontaminants, are separated into the outlet channel 137 and the wastechannels 136, respectively. The purified sample may pass through theoutlet channel 137 for further processing, or analysis. The wastechannels 136 and the outlet channel 137 may be sized and positioned toreceive a selected component. For example, the waste channels 136 arepositioned a predetermined distance from the virtual wall 15 to receivethe diffused smaller molecules and the outlet channel 137 is sized andpositioned to receive the larger molecules.

Alternatively, the configuration shown in FIG. 14 may be utilized toperform chemical manipulations, such as a chemical reaction,non-covalent binding, adsorption or absorption, antibody binding,nucleic acid or oligonucleotide binding or hybridization, ion pairing,ion exchange, chromatographic separation, receptor hormone interaction,enzyme activity antagonism or agonism, or other suitable reaction on ananalyte. The present invention applies to a variety of liquid samples,including solutions of compounds, whole cells or cell lysates, enzymes,proteins or peptides, and particles. Accordingly, the invention also hasapplications in proteomics, genomics, chromatography, diagnostics, anddrug discovery.

Alternatively, the illustrative configuration may be utilized to performa labeling operation, where the virtual wall is utilized as an interfaceport for one of the reactants in the labeling scheme. According to oneembodiment, a labeling operation is performed by running a labelingsolution through the microchannel and injecting a liquid containing asubstance to be labeled through the virtual wall. Alternatively, theliquid containing the substance to be labeled is conveyed through themicrochannel and the labeling solution is injected through a virtualwall to label the substance. The mixing of the two liquids is relativelyfast, achieving rapid labeling of the substance. According to oneembodiment, the labeling can be performed before a separation step, suchas described above. According to one embodiment, a filtration device mayalso be utilized to remove excess unreacted label after the labelingscheme.

According to another invention, a microfluidic system including avirtual wall fluid interface port also provides a device and method forpost-column detection of molecules based on their binding affinity. FIG.15 a illustrates a labeling system 1500 including a plurality of virtualwall fluid interface ports according to another illustrative embodimentof the invention. The labeling system 1500 separates a sample mixture ina column into a plurality of bands, followed by post-column labeling ofa band of one unlabeled species in the mixture that comes off the end ofa column. The system 1500 may also be used to transfer a labeled band toan analysis system, such as a MALDI-MS and/or multiwell plate basedscreening assay. As shown the labeling system 1500 includes a channel orseparation column 3. The system includes a sample injection region 1510for injecting a sample or other chemical compound into the channel orcolumn 3 and a separation region 1520 for separating the sample intobands. The system 1500 also includes a second injection region 1540formed at an end of the channel or separation column 3 for injecting aknown amount of a labeled species and/or a binding molecule into thechannel or column 3 for labeling one or more of the bands. The system1500 further includes a detection region 1550 for analyzing andidentifying compounds and an ejection region 1560, including an ejector108 for ejecting selected bands from the channel or column 3 through anejection port 17 d.

According to the illustrative embodiment, the injection regions 1510,1540 include virtual wall interface ports 17 a, 17 b, 17 c, respectivelyformed in a side wall of the channel or column 3, as described above.Other means of injecting a sample into a column are known in the art andmay be used according to the teachings of the invention. The secondinjection region 1540 may comprise two fluid interface ports 17 b, 17 chaving virtual walls formed therein are located at the end of thechannel or separation column 3, wherein the fluid interface port 17 b isused to inject the labeled species and the fluid interface port 17 c,located downstream from the port 17 b, is used to inject the bindingmolecule. Droplets of the chemical compound to be injected are formedand propelled towards the aperture 17 and enter the interior of thechannel 3 via a meniscus 15 (i.e. a virtual wall) formed by the liquidin the aperture.

The ejector 108 in the ejector region 1560 may comprise any suitableejector known in the art. Examples of suitable ejectors are describedabove with respect to FIGS. 12 a–12 g. One skilled in the art willrecognize that the ejector is not limited to the described embodimentsand that any suitable device for ejecting a sample or fraction of asample from a channel may be used.

According to an alternate embodiment, shown in FIG. 15 b, a labelingsystem 1500′ of an illustrative embodiment of the invention may includea single fluid interface port 17 b′ in the second injection region 1540′to inject the labeled species and binding molecule into the channel orcolumn 3.

Referring to FIG. 16, an illustrative embodiment of the presentinvention provides a method of labeling a sample on a microfluidiclabeling chip, such as the labeling system of FIGS. 15 a and 15 b,comprising at least one column or channel for conveying a sample andother fluids through the chip. In one application, the illustrativemethod detects a band of an un-labeled species that comes off the end ofa column. In step 1610, a sample mixture is injected into a columnthrough a first virtual wall fluid interface port 17 a, as shown indetail in FIG. 3 or through another suitable injector. In step 1620, thesample mixture is separated into bands with the on-chip column. In anoptional step 1630, the step of separating the mixture is repeated in asecond column to provide further peak discrimination. In step 1640, thebinding properties of each peak (corresponding to a separated band) areobserved by injecting label and binder pairs into the column 3 viavirtual wall fluid interface ports 17 b and/or 17 c (FIG. 15 a) and/or17 b′ (FIG. 15 b). In step 1640, a known amount of a labeled species isinjected, followed by a binding molecule, such as an antibody, whichbinds both the labeled and unlabeled species. The binding moleculeequilibrates among the bound states. The resulting bindingmolecule-labeled molecule complex (B*C) can be identified by (a) havingdifferent mobilities in the column so that the bound and unbound bandscan be observed downstream from the injection point or (b) givingdifferent Fluorescent Polarization (FP) due to the binding moleculebeing much larger than the labeled molecule or (c) other methods. Instep 1650, selected bands are chosen and ejected from the column using asuitable ejector, as described above. In step 1660, the bands aretransferred from to a MALDI-MS system and/or a multi-well plate forfurther analysis. The illustrative labeling system and method thusprovides fast transient measurement of unlabeled molecules based ontheir binding affinity.

Depending on the particular type of detection method employed, a widevariety of detectable labels may be used in applications of the presentinvention. Labels are commonly detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. The labelis coupled directly or indirectly to a molecule to be detected (aproduct, substrate, enzyme, or the like) according to methods well knownin the art, and for example, as reagents introduced onto a chip via avirtual wall formed in a side wall of a microchannel according to theillustrative embodiment of the present invention. For example, usefulnucleic acid labels include fluorescent dyes, enzymes (e.g., as commonlyused in an ELISA), biotin, dioxigenin, or haptens and proteins for whichantibodies, preferably monoclonal, are available. Other suitable labelsinclude fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Still other labeling agents include monoclonalantibodies, polyclonal antibodies, proteins, or other polymers such asaffinity matrices, carbohydrates or lipids. Detection of labeledcompounds may be by a variety of known methods, includingspectrophotometric or optical tracking of fluorescent markers, or othermethods which track a molecule based upon size, charge, molecularweight, or affinity. A detectable moiety can be of any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of gel electrophoresis, columnchromatography, spectroscopic techniques, and the like, and in general,labels useful in such methods can be applied to the present invention.Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical thermal,or chemical means; and such label may be bound covalently to themolecules of interest (e.g., reaction of amine-containing compounds withninhydrin) or non-covalently (e.g., reaction of a compound with alabeled antibody). Useful labels in the present invention includefluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), enzymes (e.g., LacZ, CAT, horse radishperoxidase, alkaline phosphatase and others, commonly used as detectableenzymes, either as marker products or as in an ELISA), nucleic acidintercalators (e.g., ethidium bromide) and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads. Fluorescent labels are particularlypreferred labels when optical detection means are employed. Preferredlabels are typically characterized by one or more of the following: highsensitivity, high stability, low background, low environmentalsensitivity and high specificity in labeling.

Several labels comprising fluorescent moieties are known, including 1-and 2-aminonaphthalene, p,p′-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin, porphyrins,triarylmethanes and flavin. In some cases, the amino acid tryptophan,which is either part of a peptide or protein of interest (i.e., it isendogenous to that peptide or protein) or which is added to said proteinor peptide, may be used as a fluorescent label. Individual fluorescentreagent compounds which may be used in accordance with the invention, orwhich can be modified to incorporate such functionalities include, e.g.,dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol;rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene;N-phenyl 2-amino-6-sulfonatonaphthalene;4-acetamido-4-isothiocyanato-stilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate;N-phenyl-N-methyl-2-aminoaphthalene-6-sulfonate; ethidium bromide;stebrine; auromine-0,2-(9′-anthroyl)palmitate; dansylphosphatidylethanolamine; N,N′-dioctadecyl oxacarbocyanine: N,N′-dihexyloxacarbocyanine; merocyanine, 4-(3′pyrenyl)stearate;d-3-aminodesoxy-equilenin; 12-(9′-anthroyl)stearate; 2-methylanthracene;9-vinylanthracene; 2,2′(vinylene-p-phenylene)bisbenzoxazole;p-bis(2-(4-methyl-5-phenyl-oxazolyl))benzene;6-dimethylamino-1,2-benzophenazin; retinol; bis(3′-aminopyridinium)1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin;chlorotetracycline;N-(7-dimethylamino4-methyl-2-oxo-3-chromenyl)maleimide;N-(p-(2-benzimidazolyl)-phenyl)maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin; rosebengal; and 2,4-diphenyl-3(2H)-furanone. Many such fluorescent labelingreagents are commercially available from SIGMA chemical company (SaintLouis, Mo.), Molecular Probes, R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology. (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City,Calif.) as well as other commercial sources known to one of skill in theart.

Fluorescent labels are one preferred class of detectable labels, in partbecause by irradiating a fluorescent label with light, one can obtain aplurality of emissions. Thus, a single label can provide for a pluralityof measurable events. Detectable signal may also be provided bychemiluminescent and bioluminescent sources. Chemiluminescent sourcesinclude a compound which becomes electronically excited by a chemicalreaction and may then emit light which serves as the detectable signalor donates energy to a fluorescent acceptor. A diverse number offamilies of compounds have been found to provide chemiluminescence undera variety or conditions. One family of compounds is2,3-dihydro-1,4-phthalazinedione. The most popular compound is luminol,which is a 5-amino compound. Other members of the family include the5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. Thesecompounds can be made to luminesce with alkaline hydrogen peroxide orcalcium hypochlorite and base. Another family of compounds is the2,4,5-triphenylimidazoles, with lophine as the common name for theparent product. Chemiluminescent analogs include para-dimethylamino and-methoxy substituents. Chemiluminescence may also be obtained withoxalates, usually oxalyl active esters, e.g., p-nitrophenyl and aperoxide, e.g., hydrogen peroxide, under basic conditions. Other usefulchemiluminescent compounds are also known and available, including-N-alkyl acridinum esters (basic H₂O₂) and dioxetanes. Alternatively,luciferins may be used in conjunction with luciferase or lucigenins toprovide bioluminescence. An illustrative example of on-chip labelingwith a fluorescent moiety may be found in Harrison, et al., Sensors andActuators B, vol. 33, pp. 105–09 (1996).

Other labeling moieties may be non-covalently bound to molecules ofinterest. Generally, a ligand molecule (e.g., biotin) is covalentlybound to a polymer. The ligand then binds to an anti-ligand (e.g.,streptavidin) molecule which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with labeled, anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody. Labels can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. Means of detecting labelsare well known to those of skill in the art. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence, e.g., by microscopy, visual inspection, via photographicfilm, by the use of electronic detectors such as digital cameras, chargecoupled devices (CCDs) or photomultipliers and phototubes, and the like.Fluorescent labels and detection techniques, particularly microscopy andspectroscopy are preferred. Similarly, enzymatic labels are detected byproviding appropriate substrates for the enzyme and detecting theresulting reaction product. Finally, simple colorimetric labels areoften detected simply by observing the color associated with the label.For example, conjugated gold often appears pink, while variousconjugated beads appear the color of the bead.

FIG. 17 illustrates a microfluidic synthesis/analysis system 40 of anembodiment of the present invention for performing a microchemicalprocess of a sample on a chip. The microfluidic synthesis/analysissystem 40 comprises a first microchannel 3, a second microchannel 30operating in parallel with the first microchannel, and a thirdmicrochannel 300 disposed at the end of the first and secondmicrochannels and forming an intersection with the first and secondmicrochannels for combining the output of the first microchannel 3 andthe second microchannel 30. The microchannels 3, 30 and 300 include aplurality of fluid interface ports 17 forming virtual walls 15 a–idisposed in the channel side walls for performing a multi-step chemicalsynthesis or analysis. The illustrative embodiment of the microfluidicsynthesis/analysis system 40 serves as an example of the application ofvirtual walls 15 in microfluidic systems in which complex reactionschemes for synthesis (e.g. labeling, as described above) and analysisare to be performed in a highly parallel fashion. As shown, a largevariety of chemical operations can be implemented by using a virtualwall 15 disposed in a sidewall 16 of a microchannel 3.

The illustrated system 40 further includes a plurality of sampleprocessors, illustrated as microreactors 43, 44, 45 and 46, disposed atselected locations in the microchannels 3, 30 and 300 for performing oneore more reactions on a sample, such as a microchemical analysis orsynthesis. According to the illustrative embodiment, the illustratedsystem is utilized to perform a separation of a sample. One skilled inthe art will recognize that any suitable process may be performed on asample, including, but not limited to a reaction, filtration, dilution,mixing, binding and transporting, alone or in combination with otherreactions. The specific arrangement shown in FIG. 17 allows performingof the following Reactions 1–4:

-   Reaction 1: A+B→C-   Reaction 2: D+E→F-   Reaction 3: C+F→G-   Reaction 4: G+H→I

In Reaction 1, substance A (e.g. a labeling reagent) reacts withsubstance B (e.g., a molecule of interest not otherwise convenientlydetectable) to form substance C (e.g a labeled conjugate). In Reaction2, substance D reacts with substance E to form substance F. In Reaction3, substances C and F react to form substance G. In Reaction 4,substances G and H react to form substance I. According to theillustrated embodiment, Reaction 1 and Reaction 2 are carried out inparallel, sequentially followed by Reaction 3 and Reaction 4.

In operation, a carrier liquid 4 a is disposed in the microfluidicsynthesis/analysis system 40 and a number liquids containing a specificchemical substance are interfaced with the carrier liquid 4 a via aplurality of virtual walls 15. The reaction products resulting fromReactions 1–4 are carried through the microfluidic synthesis/analysissystem by carrier liquid 4 a. Droplets 42 a–i are formed of liquidsrespectively containing substances A-I listed in the above reactionscheme. The overall direction of flow of the carrier liquid 4 isindicated by arrows 41. The direction of fluidic interfacing via thevirtual walls 15 is indicated by arrows 47.

To initiate the microfluidic process, a first liquid A 42 a isintroduced into the microfluidic system via a first virtual wall 15 a,as indicated by droplet direction 47, and carried through themicrochannel by the carrier liquid 4 a. A second liquid B 42 b is addedto the first microchannel 3 through a second virtual wall 15 b disposedin the side wall of the microchannel downstream from the first virtualwall 15 a, giving rise to a mixture of substances A and B present incarrier liquid 4 a. Subsequently, the mixture passes through a firstmicroreactor 43. The first microreactor 43 has appropriate conditions(i.e. temperature, residence time, presence of catalytic materials etc.)to effect Reaction 1 and produce a third substance, liquid C, from thereaction of substances A and B. Liquid C is then carried downstream fromthe reactor 43 by the carrier liquid 4 a. After completion of Reaction1, portions of liquid C are ejected from the first microchannel 3through a third virtual wall 15 c, disposed downstream of the first andsecond virtual walls 15 a, 15 c, in the form of a droplet 42 c, asindicated by droplet direction 47. The ejected portion of liquid C maybe subsequently stored, further processed or analyzed to determine thecomposition of the reacted liquid C.

In alternate embodiments of the invention, one or more of the sampleprocessors 43, 44, 45 or 46 may comprise a separation means, rather thana reaction means such as a microreactor. A separation means is typicallya chromatography column, preferably a chromatography column. Amicrofluidic system including a chromatography column may be used toperform a separation of a mixture applied, for example, via virtual wall17, and then reacted with a labeling reagent in microreactor 45 with areagent introduced through the virtual wall 15. In such a manner,compounds are labeled post-chromatographic separation. In like manner, amixture may be introduced though a first virtual wall 15 a and alabeling reagent through a second virtual wall 15 b, followed byreaction in microreactor and subsequent separation in a chromatographycolumn. Capillary electrophoresis chromatography columns areparticularly preferred separation means according to the invention. Suchmicrofluidic CE columns are described in U.S. Pat. Nos. 6,159,353,5,976,336, and 6,258,263, each of which are incorporated herein byreference. Alternatively, multiple separation means, optionally inparallel, may be employed by the present invention. For example, themicrofluidic synthesis/analysis system 40 may perform a first separationby capillary electrophoresis, and another separation based on a pHgradient.

In an alternate embodiment, a titration may be performed on-chip when acolor change occurs upon reaction. Such a titration may be, for example,a pH titration, or a titration of an enzyme with a chromogenic substrateor inhibitor.

A similar reaction process to the process that occurs in the firstmicrochannel 3 takes place in the second channel 30 of the microfluidicsynthesis/analysis system 40. Respectively, liquid D 42 d is introducedin carrier liquid 3 b via a fourth virtual wall 15 d. Downstream fromthe injection point for liquid D (i.e. virtual wall 15 d), liquid E 42 eis injected into the microchannel 3 via a fifth virtual wall 17 e toform a mixture of substances D and E in the carrier liquid 4 a. Furtherdownstream, the mixture of substances D and E passes through a secondmicroreactor 44, wherein Reaction 2 proceeds. After completion ofReaction 2, a portion of the resulting liquid F can be ejected from thesecond microchannel 30 via a sixth virtual wall 15 f. The ejectedportion of liquid F may be subsequently stored, further processed oranalyzed to determine the composition of the reacted liquid F.

At the point of intersection of the first microchannel 3 and the secondmicrochannel 30, the liquid C (42 c) leaving the first microreactor 43and the liquid F (42 f) leaving the second microreactor 44 mix togetherand are subsequently carried through the third microchannel 300. Themixture of liquid F and liquid C enters the third microreactor 45, inwhich Reaction 3 proceeds and produces liquid G. A seventh virtual wall15 g disposed downstream from the third microreactor to allow forportions of liquid G leaving the third microreactor 45 to be ejectedfrom microchannel 3 in the form of liquid droplets 42 g. An eighthvirtual wall 15 h disposed further downstream from the seventh virtualwall liquid 15 g is utilized to introduce substance H into themicrochannel 300 in the form of liquid droplet 42 h, resulting in amixture of liquid G and liquid H. The G-H mixture enters a fourthmicroreactor 46, in which Reaction 4 ensues to form liquid I. Finally,portions of the resulting liquid I 42 i leaving the fourth microreactor46 are ejected from the microchannel 3 via a ninth virtual wall 15 idisposed in the sidewall of the microchannel 300 for analysis, storageand/or further processing of the liquid I.

According to the illustrative embodiment, the required concentrations ofreactants A, B, D, E and H are precisely controlled by metering the sizeand number of droplets (19 b). Moreover, by controlling the number ofdroplet 19 b introduced in carrier liquid 4 a, a specific dilution canprecisely be obtained allowing the study of reactions 1–4 for differentdilutions of reactants A, B, D, E and H. A suitable droplet dispensingsystem for forming droplets of suitable size is described in ProvisionalU.S. Patent Application 60/325,040. The use of a direct interface portformed by a virtual wall in the sidewall of the microchannel in theillustrative microfluidic synthesis/analysis system 40 allows precisecontrol over the concentrations of liquids that are introduced into thesystem and significantly reduces waste while increasing efficiency.

The illustrative embodiment the microfluidic synthesis/analysis system40 described above serves as an example of the application of a virtualwall 15 in microfluidic systems in which complex reaction schemes forsynthesis and analysis are to be performed in a highly parallel fashion.As shown, a large variety of chemical operations can be implemented byusing virtual wall 15 disposed in a sidewall of a microchannel. Oneskilled in the art will recognize that the invention is not limited tothe illustrative embodiment and that any suitable size and number ofmicrochannels, virtual walls, reactor types and numbers and sample typesmay be utilized in accordance with the teachings of the invention.

FIG. 18 illustrates an alternate embodiment of the microfluidic systemof the present invention. The system 10 includes a microchannel 3including one or more fluid interface ports forming a virtual wall 15and comprises a part of an electrokinetically operated system 22. In theelectrokinetically operated system 22, an electric field is establishedto transport fluid through the microchannel 3. As illustrated a firstelectrode 5 and a second electrode 7 are disposed in or in fluidcommunication with the first liquid 4 in the microchannel 3. The firstelectrode 5 is disposed in a first well 6 a located at a first end ofthe microchannel 3 and the second electrode 7 is disposed in a secondwell 6 b located at a second end of the microchannel 3. The first well 6a and the second well 6 b are in fluid communication with the firstliquid 4 in the microchannel 3. A voltage generator 12 generates avoltage between the first electrode 5 and the second electrode 7, whichproduces a substantially longitudinal electric field in the first liquid4 in the microchannel 3. As shown in FIG. 18, the longitudinal electricfield is applied by bringing both ends of microchannel 3 in fluidiccontact respectively with a first well 4 and a second well 6.

The illustrated port 17 is disposed in the sidewall of the microchannel3 and forms a virtual wall 15 allowing the introduction of a secondliquid into a first liquid 4, according to the teachings of theinvention. For example, the droplet generating system 18 forms a droplet19 a of the second fluid 19 b. The droplet 19 a is directed towards theport 17 via any suitable method, such as those described herein.Depending on the surface properties of the inner walls of themicrochannel 3, the longitudinal electric field generated by theelectrokinetic system 22 induces an electroosmotic liquid flow of thefirst liquid 4 in an axial direction through the microchannel 3, therebysubstantially transporting the constituents present in the second liquid19 b in the axial direction through the microchannel.

The electrokinetically operated system 22 can include an optionaldetector 68 to provide for electrophoretic analysis of the constituentspresent in the second liquid 19 b. The opening 17 disposed in themicrochannel 3 has suitable properties, such as a selected diameter,length, and inner wall surface properties, to provide electrophoreticseparation of the constituents of a liquid. A second liquid 19 b to beanalyzed is introduced to the microchannel 3 via the virtual wall 15 inthe form of a droplet 19 a generated by a droplet generating system 18.Constituents present in the introduced second liquid 19 b aretransported by a combination of electroosmotic flow and migration underthe influence of the electric field applied with the voltage generator12. After traveling a sufficient distance to result in electrophoreticseparation of the liquid, the detector 68 detects and analyzes theindividual constituents at the end of microchannel 3 by the detector 68.The detector 68 generates an electropherogram from which the compositionof second liquid 19 a can be determined. Those of ordinary skill willunderstand that the electrokinetic system of the invention can performelectroosmotic, electrophoretic, and dielectrophoretic techniques.

FIG. 19 illustrates another implementation of an electrokineticallyoperated microfluidic system 50 according to the present inventioncomprising a plurality of parallel microchannels 3 a–3 h formed in acartridge 73 in which a substrate 20 is disposed. The microchannels areformed by forming a network of half-open channel structures in thesubstrate and covering the half-open channels with a cover 20 a to formthe plurality of microchannels. The plurality of microchannels 3 a–3 hare disposed in the substrate in communication with a common first well6 a and a common second well 6 b. The microchannels 3 a–h include one ormore fluid interface ports 17 defined by openings formed in the channelside wall that are sized and dimensioned to form virtual walls when themicrochannels are filled with a first liquid 4. A first electrode 5connected to a voltage generator 12 is disposed in the common first well6 a and a second electrode connected to the voltage generator 12 isdisposed in the common second well 6 b to establish a substantiallylongitudinal electric field in the microchannel 3. Droplets 19 b aregenerated by a droplet generator 18. The droplet generator 18 forms andpropels the droplets towards a selected virtual wall 15 formed in afluid interface port form in the side wall of a selected microchannel ina suitable direction and with a suitable velocity to introduce thedroplet to the selected microchannel 3 via the opening 17. A detector 68is positioned relative to the system 50 to monitor and detect the fluidin the microchannels 3 a–3 h.

In a preferred embodiment, the parallel implementation of anelectrokinetically operated system 50 is applied as a highly parallelelectrophoretic separation platform, capable of performing a largenumber of analyses per unit of time. The microchannels 3 disposed in thesubstrate 20 have suitable properties for electrophoretic separation(inner wall surface properties, diameter and length). The process ofelectrophoretic analysis of the constituents of droplet 19 b isidentical as described before. The electrokinetically operated apparatus50 further includes a detector 68 for detecting and analyzing theindividual constituents at the end of microchannels 3. The detector 68generates an electropherogram from which the composition of secondliquid 19 a can be determined.

As shown, the illustrative electrokinetically operated system 50comprises a compact structure, which allows a plurality of differentreactions and processes to occur on a relatively small substrate 20. Theuse of openings forming virtual walls to define fluid interface ports inthe side walls of the parallel microchannels 3 a–3 h allows directinterfacing of fluid with the microchannels, improves injectionefficiency and provides easy control over the volume of liquidintroduced into the system 50.

According to yet another application, the virtual walls may be utilizedto interface a microfluidic system 181 with a mass spectrometer, asshown in FIG. 20. For example, a sample may be injected into amicrochannel 3 via a virtual wall 15 according to the teachings of theinvention and the sample may then be separated into differentcomponents. After separation, the different components may be ejectedfrom the microchannel through a virtual wall 15 forming a fluid ejectionport in the form of droplets. The droplets may be directed onto asuitable plate 185 for analysis with a mass spectrometer 186 or otherrobotic system. For example, the microfluidic system 181 may inject theseparated samples onto a multi-well plate to form a multi-well array ofthe sample for analysis.

FIGS. 21 a, 21 b and 21 c illustrate a method of manufacturing amicrofluidic chip 190 having virtual wall interface ports for processingliquid samples. FIG. 21 a is an exploded view of a microfluidic chip 190employing virtual wall fluid interface ports according to the teachingsof the invention. FIG. 21 b is a top view of the microfluidic chip 190of FIG. 21 a. FIG. 21 c is a cross-sectional side view of themanufactured microfluidic chip 190 of FIG. 21 a. As shown, amicrochannel 3 with a virtual wall fluid interface ports 17 formed inthe side wall of the microchannel may be manufactured with a three-layer“sandwich” construction. As shown, a complete microfluidic chip 190having a microchannel 3, a first inlet 191 formed at a first end of themicrochannel 3, an outlet 192 formed at a second end of the microchannel3, and a fluid interface port 17 formed in a side wall along the lengthof the microchannel 3. The microfluidic chip shown in FIGS. 21 a–ccomprises a first planar sheet 193 having a recess 194 formed therein, amiddle layer 197 including a channel 3 and an opening 17 and a secondplanar sheet 195. The opening 17 has dimensions between about 0.1 μm andabout 200 μm and preferably between about 25 μm and about 125 μm andmost preferably between about 50 μm and about 100 μm, so that a liquiddisposed in the microchannel forms a virtual wall at the opening 17.

To manufacture the microfluidic chip 190, a portion of the first planarsheet 193 is removed to form the recess 194. Next, the middle layer isapplied on top of the first sheet. Then, a portion of the middle layer197 is removed to form the opening 17. Additional portions of the middlelayer 197 are removed to form slits defining the microchannel, which arealigned with the recess. The second planar sheet 195 is applied to themiddle layer 197, and a virtual wall access hole 170 is formed in thesecond planar sheet 195 prior to application or after application of thesecond planar sheet to the middle layer by removing a portion of thesecond planar sheet. The virtual wall access hole 170 is aligned withthe opening 17 formed in the middle layer to provide access to themicrochannel. A low temperature bonding process may be utilized toassemble the three layers forming the chip.

According to the illustrative embodiment, the first and second planarsheets 193 and 195 comprise glass plates, though one skilled in the artwill recognize that any suitable material may be used. Etching,powderblasting, or any suitable method may form the recess 197, channel3, inlet 191, outlet 192 and the openings 17 and 170 forming the fluidinterface port. According to an alternate embodiment, the recess 197,channel 3, inlet 191, outlet 192 and the openings 17 and 170 may bepre-formed in the layers, i.e. the layers may be molded to form thestructures. According to the illustrative embodiment, the middle layer197 is comprised of a photo patternable material, such as aphotosensitive polymer applied by lamination. According to an alternateembodiment, the middle layer 197 of the microfluidic chip 190 alsocomprises a glass plate.

The use of glass plates to form the microfluidic chip 190 yields betteroptical detection of the microchannel 3 interior, as the top and bottomof the microchannel are formed by two parallel glass surfaces. Inconventional round capillaries and also in etched channels on chip (alsopartly round surfaces) there is substantial light scattering due to thecurved surfaces through which the light should pass. The glass surfacesare very flat and smooth to enhance the flow of fluid through themicrochannel 3, and further facilitate and enhance electrophoresis.Furthermore, using glass substrates significantly reduces the cost ofmanufacturing the microfluidic chip 190.

According to one embodiment, a plurality of microchannels may formed inthe microfluidic chip, and can be configured to intersect with eachother.

FIGS. 22 a–c illustrate the steps of manufacturing a microchannel havinga fluid interface port 17 defined by an opening suitable for forming avirtual wall according to an embodiment of the invention. FIG. 22 a is across-sectional view of a substrate 28 in which a first portion of anopen channel 29 is formed. To form an enclosed microchannel, thesubstrate 28 is then covered with a cover 30, as shown in FIG. 22 b.Subsequently, at least a portion of the cover 30 is removed to form thefluid interface port 17 in microchannel 3, as shown in FIG. 22 c. Asdiscussed, the fluid interface port 17 is sized and dimensioned to forma virtual wall when the microchannel is filled with a first liquid. Inanother embodiment the fluid interface port 17 is disposed in the cover30 prior to bonding the cover 30 on top of substrate 28.

According to the illustrative embodiment, the substrate 30 and the cover28 are formed of silicon, though one skilled in the art will recognizethat any suitable material for forming a microchannel 3 in amicrofluidic device or system may be utilized. For example, themicrofluidic system may be made out of glass, plastic or any othersuitable material. The microchannel 3 may be fabricated from a siliconwafer substrate 30 using a standard photolithography etching process tofabricate the microchannel structures. A photolithography process mayalso be utilized to etch the fluid interface port 17 in the cover 28.One skilled in the art will recognize that alternative materials andmanufacturing techniques, such as wet chemical etching, controlled vapordeposition, laser drilling, and the like, may be utilized.

FIGS. 23 a–23 c illustrate the steps of manufacturing a microchannelwith a virtual wall interface port according to another embodiment ofthe invention. FIG. 23 a is a cutaway view of a substrate 28 in which ahalf open channel structure 29 is disposed. The substrate 28 is thencovered with a first cover 30 a on which a second cover 30 b isdisposed, as shown in FIG. 23 b to form the enclosed microchannel 3.Subsequently, at least a portion of the first cover 30 a and a portionof the second cover 30 b are removed as to form the fluid interface port17 in microchannel 3. As shown, the opening extends through the firstcover 30 a and the second cover 30 b to form a fluidic interface betweenthe interior of the microchannel 3 and the exterior of the microchannel.In another embodiment the fluid interface 17 is formed in first cover 30a and second cover 30 b prior to bonding of said first and second cover30 a and 30 b to the top of the substrate 28.

According to the illustrative embodiment, the substrate 30 and the firstand second covers 30 a and 30 b are formed of silicon, though oneskilled in the art will recognize that any suitable material for forminga microchannel 3 in a microfluidic device or system may be utilized. Forexample, the microfluidic system may be made out of glass, plastic orany other suitable material. The microchannel 3 may be fabricated from asilicon wafer substrate 30 using a standard photolithography etchingprocess to fabricate the microchannel structures. A photolithographyprocess is utilized to etch the fluid interface port 17 in the first andsecond covers 30 a and 30 b. One skilled in the art will recognize thatalternative materials and manufacturing techniques, such as wet chemicaletching, controlled vapor deposition, laser drilling, and the like, maybe utilized.

FIGS. 24 a–24 c illustrate the steps of manufacturing a microchannelhaving a virtual wall as a fluidic interface port according to analternate embodiment of the invention. In the embodiment shown in FIGS.24 a–24 c, the opening forming the virtual wall is formed in a substratehaving a half-open channel structure, rather than a cover for enclosingthe half-open channel structure. FIG. 24 a shows an exploded view of asubstantially planar first cartridge part 35 in which a half openchannel structure 37 is disposed. A portion of the side wall of isremoved from the half open channel structure 37 in the substrate 35 toform a fluid interface port 17. A second cartridge part 36 is bonded tothe top of the first cartridge part 35 to define a microchannel 3.

According to the illustrative embodiment, the first cartridge part 35and the second cartridge part 36 are formed of silicon, though oneskilled in the art will recognize that any suitable material for forminga microchannel 3 in a microfluidic device or system may be utilized. Forexample, the microfluidic system may be made out of glass, plastic orany other suitable material. The microchannel 3 may be fabricated in thefirst cartridge part 36 using a standard photolithography etchingprocess. A photolithography process is utilized to etch the fluidinterface port 17 in the side wall of the first cartridge part. Oneskilled in the art will recognize that alternative materials andmanufacturing techniques, such as wet chemical etching, controlled vapordeposition, laser drilling, and the like, may be utilized according tothe teachings of the present invention.

FIG. 24 b and FIG. 24 c show respectively a perspective view and acutaway view of the resulting virtual wall 15 formed in themicrochannel. As illustrated, the fluid interface port 17 formed in themicrochannel is sized and dimensioned to form a virtual wall at theopening 17 when the microchannel is filled with a first liquid 4. Thevirtual wall 15 allows fluidic interfacing with the microchannel asdescribed above and is sized and dimensioned to retain liquid within themicrochannel without adversely affecting liquid flow through themicrochannel. FIGS. 21 a and 21 c illustrate the introduction of adroplet 19 b of a second liquid 19 a into a first liquid 4 present inthe microchannel 3 via the virtual wall 15 formed within the opening 17.As shown, the droplet is propelled towards the virtual wall andtraverses the virtual wall to enter the microchannel.

FIGS. 25 a and 25 b illustrate an alternate embodiment of themicrochannel system of FIGS. 21 a–21 c. FIG. 25 a shows an exploded viewof a substantially planar first cartridge part 35 in which a half openchannel structure 37 is disposed. A portion of the side wall of thecartridge part 35 is removed from the half open channel structure 37 toform an opening 17. A hydrophobic path 38 is disposed substantiallyopposite the opening 17 in the half open channel structure 37. A secondcartridge part 36 is bonded to the top of the first cartridge part todefine the microchannel 3. Upon filling of the microchannel 3, a firstvirtual wall 15 is formed at the opening 17 and a second virtual wall 15a is defined by the hydrophobic patch 38.

The hydrophobic patch enhances introduction of a second liquid into themicrochannel. The hydrophobic patch 38 attracts first fluid 4, andprevents escape of the fluid through virtual wall. As illustrated inFIG. 25 b, a droplet of a second liquid is introduced into themicrochannel via the virtual wall and drawn into the microchannel by thehydrophobic patch.

The use of a virtual wall in a microchannel side wall to create abi-directional fluid interface for a microfluidic system providessignificant advantages over conventional fluid interfaces. The fluidinterface port comprising a virtual wall is relatively simple tomanufacture, is compact, provides high ejection efficiency, does notadversely affect operation/flow, can be made bi-directional and isuseful for a variety of applications. The illustrative embodimenteliminates the need for a separate structure, such as a channel or areservoir and permits direct injection of a sample into a microchannel.

EXEMPLIFICATION OF THE INVENTION Example 1 Virtual Wall MicrofluidicChip Manufacturing and Use

Microchannel structures having a fluid interface port were manufacturedby isotropic etching half-open channels, 100 micrometer in width, 50micrometer in height and a length of 20 mm, in a 1.1 mm thick glasswafer. A buffered hydrogen fluoride (HF) solution was used as an etchantand photo patterned etchant resistant silicon nitride mask layers wereapplied to define the microchannel areas to be etched. Access to thechannels was provided by powder blasting 1-mm diameter holes completelythrough the glass wafer at both ends of each etched half-openmicrochannel.

A covering 50 micrometer thick layer of dry resist film (LAMINAR® 5000,Shipley, Birkenfeld, Germany) was applied on top of the etched wafer.Fluid interface ports were incorporated by photo patterning circularapertures of 50–150 micrometer in the dry resist film whereby theapertures were aligned with and extended into the underlyingmicrochannels. Finally the wafer was diced in individual chips, whichwere placed in a holder to connect the microchannel structures toexternal systems, such as fluid pumps and electric power supplies.

The resulting microchannels were filled with an aqueous buffer solution(50 mM bi-carbonate buffer, pH 9.0). It was observed that themicrochannels filled automatically via capillary forces on theapplication of liquid to the fluid interface port. Liquid drops having adiameter of about 50 micrometer were produced using apiezoelectric-actuated drop dispenser system (MicroDrop GmbH,Norderstedt, Germany). Drops were formed from a buffered solutioncontaining a fluorescent dye (50 mM bi-carbonate buffer, pH 9.0, 1 mMfluoresceine) and were directed to a 100 micrometer diameter interfaceport. The injection process was monitored using an inverted microscopeunder UV light irradiation to excite the fluorescent dye and obtain abright yellow-green color.

The present invention has been described relative to an illustrativeembodiment. Since certain changes may be made in the above constructionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description or shown in theaccompanying drawings be interpreted as illustrative and not in alimiting sense. It is also to be understood that the following claimsare to cover all generic and specific features of the inventiondescribed herein, and all statements of the scope of the inventionwhich, as a matter of language, might be said to fall therebetween.

1. A method of labeling a sample, comprising: conveying the samplethrough a channel having a first virtual wall fluid interface port, thefirst virtual wall fluid interface port comprising a first openingformed in a side wall of the channel, the first opening sized anddimensioned such that a fluid within the channel forms a virtual wall inthe first opening to define the first virtual wall fluid interface port;separating the sample in the channel into a plurality of bands; andinjecting a labeling solution through the first virtual wall fluidinterface port, wherein the labeling solution interacts with one of saidbands to form a labeled band.
 2. The method of claim 1, furthercomprising the step of detecting the labeled band.
 3. The method ofclaim 1, further comprising the step of ejecting at least a portion ofthe labeled band from the channel.
 4. The method of claim 3, wherein thestep of ejecting a portion of the labeled band comprises ejecting atleast a portion of the labeled band through a second virtual wallinterface port in the form of one or more droplets, the second virtualwall fluid interface port comprising a second opening formed in a sidewall of the channel, the second opening sized and dimensioned such thata fluid within the channel forms a virtual wall in the second opening todefine the second virtual wall fluid interface port.
 5. The method ofclaim 1, wherein the labeling solution comprises a labeled species and abinding molecule for binding to a selected band and the labeled species.6. The method of claim 5, wherein the step of injecting the labelingsolution comprises injecting the labeled species through the firstvirtual wall fluid interface port and injecting the binding moleculethrough a second virtual wall fluid interface port, the second virtualwall fluid interface port comprising a second opening formed in a sidewall of the channel, the second opening sized and dimensioned such thata fluid within the channel forms a virtual wall in the second opening todefine the second virtual wall fluid interface port.
 7. The method ofclaim 1, wherein the virtual wall fluid interface port has a diameterbetween about 25 μm and about 100 μm, such that when a fluid is disposedin the interior of the channel, the fluid forms a virtual wall at thevirtual wall fluid interface port.
 8. The method of claim 1, furthercomprising the step of separating the plurality of bands in to aplurality of sub-bands.
 9. The method of claim 1, further comprising thestep of transferring a labeled band to one of a MALDI-MS system and amulti-well plate for further analysis.
 10. A method of labeling asample, comprising: conveying the sample through a channel having avirtual wall fluid interface port, the virtual wall fluid interface portcomprising an opening formed in a side wall of the channel, the openingsized and dimensioned such that a fluid within the channel forms avirtual wall in the opening to define the virtual wall fluid interfaceport; and injecting a labeling solution through the virtual wall fluidinterface port, wherein the labeling solution interacts with the sampleto label the sample.
 11. The method of claim 10, further comprising thestep of separating the sample into a plurality of bands.
 12. The methodof claim 4, wherein the step of ejecting employs a pressure pulsegenerator, said pressure pulse generator comprising a conical-shapedpressure chamber disposed opposite the second virtual wall fluidinterface port and in communication with the interior of the channel anda piezo-actuated membrane connected to the pressure chamber.
 13. Themethod of claim 4, wherein the step of ejecting employs a pin assemblycomprising a first pin and a second pin spaced from the first pin todefine a capillary for receiving a liquid volume.